The emergence of a mature nervous system requires a significant refinement of the synaptic connections initially formed during development. Redundant synaptic connections are removed in a process known as synapse elimination. Synapse elimination has been extensively studied at the rodent neuromuscular junction (NMJ). Although several axons initially converge onto each postsynaptic muscle fiber, all redundant inputs are removed during early postnatal development until a single motor neuron innervates each NMJ. Neuronal activity as well as synaptic glia influence the course of synapse elimination. It is, however, unclear whether target muscle fibers are more than naïve substrates in this process. I examined the influence of target myofiber contractile properties on synapse elimination. The timing of redundant input removal in muscles examined correlates strongly with their proportion of slow myofibers: muscles with more slow fibers undergo elimination more slowly. Moreover, this intermuscular difference in the timing of synapse elimination appears to result from local differences in the rate of elimination on fast versus slow myofibers. These results, therefore, imply that differences in the constituent fiber types help account for the variation in the timing of the developmental synapse elimination between muscles and show that the muscle plays a role in the process. In the central nervous system (CNS), pruning contributes to learning and memory4. The importance of proper synaptic maturation is highlighted by the growing body of literature that implicates defects in developmental CNS synapse elimination in the genesis of debilitating neurodevelopmental disorders6.Synaptic connections – and the neuronal networks which they form – undergo significant transformation during maturation of the nervous system. Such maturation is essential for proper architecture and function of the nervous system. This synaptic transformation includes synapse elimination, a process in which multiple immature presynaptic inputs converge at and compete for control of a common postsynaptic target. Similar pruning occurs throughout developing nervous systems9. Synapse elimination at developing endplates, like elsewhere in the nervous system13, is influenced by activity. The relative levels and the pattern of activity influence the winner and the timing with which the competition amongst immature axonal inputs that converge at developing endplates is resolved17. In addition, recent studies also implicate active glial participation in the appropriate and timely pruning of excess axonal inputs21. Little is known about what instructive role, if any, the postsynaptic targets play in synapse elimination.Although widespread throughout the developing nervous system, synapse elimination is most extensively studied and perhaps best understood at developing rodent neuromuscular junctions (NMJs), cholinergic synaptic connections between axon terminals of spinal motor neurons and their target skeletal muscle fibers. At each mature NMJ, the presynaptic terminals of a single motor neuron appose high-density aggregates of postsynaptic acetylcholine receptors (AChRs), and the processes of terminal Schwann cells (tSCs) cap the synaptic apposition. In rodents, this is achieved within the first three postnatal weeks by locally pruning all but one of up to ~10 motor axons that converge onto a postsynaptic muscle fiber23. Tonic fibers are common in amphibians and reptiles but found only in a subset of extraocular muscles in mammals29. In all species examined, these tonic fibers remain innervated by multiple motor axons into adulthood33. In contrast, the twitch fibers achieve single innervation. The twitch fibers are further divided, crudely, into two sub-classes based on their contraction speeds: slow twitch (type I) and fast twitch (type II)34. Such divergence between tonic and twitch fibers, in their ability and/or need to tolerate multiple motor innervation, hints that properties of these two distinct twitch fibers types (contractile or otherwise) may also differentially influence excess motor input removal.Based on the two fundamentally different types of contractions they produce, skeletal muscle fibers can be classified either as “tonic”  or “twitch” 39). Cross-innervation experiments – in which muscles are experimentally innervated by foreign nerves that normally innervate muscle of different fiber type – clearly show muscle fiber types are sensitive to the activity patterns presented via the innervating motor axons. These fibers alter their contractile properties to match the firing patterns of the axons present in the foreign nerve41. The normal differentiation of fiber types and their normal intramuscular distribution, however, can occur even in the absence of innervation42. Thus, it may be difficult to discern whether fiber type-specific differences in synapse elimination stem from muscle-autonomous influences and/or as nerve-driven changes to the postsynaptic fiber types.The interaction between the nerve and muscle at NMJs presents an added layer of complexity in determining the contributions of each during synapse elimination. The contractile properties of the slow and fast fibers match the activity patterns of their respective presynaptic motor neurons. The presynaptic nerve terminals and the target muscle fibers influence each other via an exchange of signals known to occur at NMJs  and those with muscle-specific expression of Cre recombinase under the human skeletal actin promoter . The generation and initial characterization of genetically modified mouse lines used in this study were described previously45. Mice were genotyped by PCR using the following primers:Experiments were conducted in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committees at Texas A&M University. The intramuscular comparison of synapse elimination for NMJs situated on slow and fast fibers was performed in wildtype C57BL/6 mice. All mutants and transgenic animals utilized have the same C57BL/6 genetic background. The transgenic overexpression of PGC1α was achieved under a muscle-specific muscle creatine kinase promoter . Mice whose skeletal muscle fibers lacked PGC1α were generated by breeding animals that harbor floxed PGC1α allele . For fluorescence imaging of muscle whole mounts, euthanized animals were transcardially perfused with PBS, pH 7.4. The sternomastoid, triangularis sterni, extensor digitorum longus (EDL), plantaris and soleus muscles were dissected and fixed in 4% phosphate-buffered paraformaldehyde, pH 7.4 for 20 minutes at room temperature and rinsed in three changes, 5 min. each, of PBS.For muscle fiber type determination, fixed sternomastoid, EDL and soleus muscles were and frozen in Tissue-Tek Optimal Cutting Temperature compound  with liquid nitrogen-cooled isopentane. The blocks of muscles were cut on a cryostat  to produce 12-µm sections at an angle perpendicular to the long axis of the muscle fibers.48. Briefly, the initially ovoid plaques of AChR aggregates morph successively into perforated plaques and open configurations.To label surface nicotinic AChR at NMJs, fixed muscles were incubated with α-bungarotoxin  conjugated to spectral variants of Alexa Fluor fluorescent dyes  prior to permeabilization (0.1 mg/ml). The motor axons were labeled with a monoclonal anti-neurofilament antibody . If an NMJ is innervated by a single motor axon Fig.  arrow, tType I muscle fibers were labeled with one of two monoclonal anti-slow myosin heavy chain antibodies: anti-MHCs (Leica Biosystems), and A4.840 . The specificity of the Leica anti-MHCs monoclonal  was verified by co-labeling of muscle cross-sections with A4.840  and isotype-specific anti-mouse secondary antibodies conjugated to spectrally distinct fluorescent dyes Fig. . A rabbi49.Images were acquired using a Zeiss LSM 780 confocal system  or a Leica DMR epifluorescence microscope equipped with a Hamamatsu cooled CCD camera. Confocal images were acquired using a 40X oil-immersion objective (N/A 1.4) with a 212.55 µm × 212.55 µm field of view at 2048 × 2048 pixel-density and 0.3 µm steps in the Z-axis. Analysis of digital images and determination of single- vs. polyneuronal innervation were performed using FIJI softwareStatistical analyses – one-way analysis of variance (ANOVA) with Bonferroni post hoc, linear regression, Student’s t-test, and Pearson’s/Spearman correlation coefficients – of raw data and the generation of histograms were performed using GraphPad Prism software  and Excel spreadsheet software : *p < 0.05; **p < 0.01; ***p < 0.001. Numerical data are reported as mean ± standard error of means (SEM).51. The timing of developmental synapse elimination in various skeletal muscles also seems to generally follow this AP developmental gradient52. Consistent with this observation, I have previously observed that the completion of neuromuscular synapse elimination in a neck muscle, the sternomastoid (STM), occurs several days prior to that in a hindlimb muscle, the soleus (SOL)53. Those results are confirmed in the present study. Additional influences independent of the AP axis, nevertheless, exert influence on the removal of redundant motor inputs59.Progressive development along the anterior-posterior  axis occurs for many aspects of mammalian neuromuscular development, including initial differentiation of motor neurons and muscles and the generation of functional neuromuscular connections and reflex circuits60). Counter to the AP axis-based prediction, I found that roughly half of the NMJs – identified with fluorescently-labeled α-bungarotoxin (α-BTX) – in SOL and TS are innervated by multiple axons at postnatal day (P) 9, while this was true for only ~15% of STM NMJs . The thinness of TS proved prohibitive in determining the slow muscle fiber contribution to its makeup via production of cross-sections. Based on whole-mount images . Lastly, the present findings appear consistent with another previous study in which delayed synapse elimination is concurrent with significantly fewer type II fibers in muscles of a spinal muscular atrophy mouse model, SMAΔ753. Together, these data support the possibility that muscles with greater fractions of slow fibers  are slower to undergo synapse elimination versus muscles composed primarily of fast fibers .To control for the influence of AP positioning of the muscles examined, I compared SOL (rich in slow-twitch myofibers) with two fast twitch-rich hindlimb muscles extensor digitorum longus (EDL) and plantaris (PLT)Next, I tested whether experimentally manipulating fiber type composition of a muscle would correspondingly affect the timing of synapse elimination. Soleus muscle offers several advantages for examining the potential influence of muscle fiber type on developmental synapse elimination. Firstly, it has significant proportions of both slow and fast fibers Figs , 2A,B – 43. Second, transgenic mice that overexpress PGC1α under a myofiber-specific promoter (MCK-PGC1α) have been reported to exhibit increased proportions of slow fibers44. I confirmed a modest – yet consistent – reduction in slow fibers in the soleus muscles of PGC1α-MKO animals, but failed to detect an increase in slow fiber frequency in SOL of MCK-PGC1α transgenic mice  produce altered muscle fiber type compositions. First, I utilized mutant mice that harbor myofiber-specific inactivation of PGC1α (PGC1α-MKO); this mouse line exhibits a reduced proportion of constituent slow muscle fibersfl/fl; 56.37 ± 0.38%, n = 4, p = 0.1476). The values for the two control groups (wildtype and PGC1αfl/fl) were pooled for further statistical analysis. At P9, PGC1α-MKO SOL had ~15% fewer polyneuronally innervated NMJs compared to those of control pups while no difference was seen between control and MCK-PGC1α  or muscle-specific expression of the Cre-recombinase . A decrease in physical activity and muscle function were reported for PGC1α MKO mice43. It is, however, unclear whether these changes stem from changes to muscle physiology and/or motor neuron activity. Even if the decrease in physical activity were due to changes in motor neuron activity, the overall decrease in neuromuscular activity is predicted to delay the timing of synapse elimination64, the opposite of what was observed with the PGC1α muscle knock-out pups  and those homozygous for the floxed PGC1α allele  where the slope of the best-fit line (1.452 ± 0.123) deviated significantly from zero  using values already at hand Figs . The anaThe above findings also raise the possibility that developmental synapse elimination occurs at different rates for NMJs situated on fibers with distinct contractile properties. It is, however, equally possible that altered fiber type compositions alter the milieu of a given muscle as a whole, within which the elimination of surplus motoneuron inputs on all synapses are similarly influenced. In an attempt to distinguish between the two possibilities, I compared the proportions of SOL NMJs situated on slow and fast fibers that receive multiple innervations. To determine unambiguously the fiber type associated with each NMJ, I employed confocal microscopy that allows examination of each synapse in all three spatial dimensions . Most importantly, the AChR maturation score bears little correlation to the fiber type of the myofiber on which they are found  and the proportions of polyneuronally innervated NMJs at different stages of postsynaptic maturation do not significantly differ  and converging motor axon terminals are known to affect the timing and/or the winner of this competition20. Secondly, while virtually all SOL NMJs are polyneuronally innervated at P3, the fractions of NMJs still polyneuronally innervated at P9 differ between types I and II muscle fibers. A recent report7 demonstrated that motor axons initially establish synaptic connections, albeit weak, with nearly all available NMJs within a given target muscle. This observation suggests that the number of axons that initially converge at a typical neonatal endplate does not differ between types I and II muscle fibers. Together, these findings suggest that the differences in the timing of resolution of synapse elimination between muscles and distinct fiber types within a given muscle both likely stem, at least partly, from differences in the rate of synapse elimination of distinct fiber types.The present findings, taken together with previous reports, may suggest that the rates of synapse elimination indeed differ between muscles and between fiber types. Firstly, I have previously reported that while virtually all NMJs of STM and SOL are polyneuronally innervated 3 days after birth, a progressively smaller fraction of STM NMJs compared to SOL remain polyneuronally innervated through postnatal days 6 and 974. Moreover, skeletal muscles could be assigned either “Fast” or “Delayed” synapsing with respect to the initial formation of NMJs and subsequent synaptic stability upon denervation75. Had synapse elimination been completed in the order in which the muscle fibers are generated, slow fibers would be expected to reach single innervation before fast fibers. Similarly, if the order in which NMJs form dictates the timing of redundant motor input pruning, the expected outcome where “Fast” synapsing muscles – including EDL – outpace “Delayed” synapsing muscles (PLT and SOL) would deviate from my present observations. The results of this study, in contrast, strongly favor the idea that properties of individual myofibers, perhaps more so than the order of generation of a fiber or its resident synapse, influence the developmental pruning of surplus motor inputs. Indeed, a linear regression analysis of my present data suggests that differences in the abundance of slow fibers account for a significant fraction of the variability in the timing of synapse elimination amongst the muscles examined.In addition to the progression of differentiation down the AP axis, there are developmental programs that appear to influence the generation of the neuromuscular system and potentially impact the timing of synapse elimination. Muscle fibers generated during the first wave of myogenesis are reported to be primarily of slow contracting type while a majority of those formed during secondary myogenesis become fast contracting17. Such stimulation also produced a shift in contractile properties of SOL – a “slow” muscle – to resemble a “fast” muscle including more rapid rise times for isometric twitches, a requirement of higher stimulation frequency for fusion of twitches, and larger tetanus contractions despite smaller isometric twitches. While such direct stimulation may also activate preterminal axons or neuromuscular junctions in addition to the muscle fibers, the concurrent changes in contractile properties consistent with slow-to-fast fiber type conversion and precocious synapse elimination are consistent with my current findings where removal of excess motor inputs lags on slow type muscle fibers. Retention of multiple innervation by tonic muscle fibers – those that do not generate action potentials – in reptiles31 may also suggest that properties of target muscle fibers can influence synapse elimination. The observed difference in the timing of synapse elimination between twitch fiber types is, then, perhaps not unexpected.The patterns in which muscles are stimulated to contract is known to influence the time course of synapse elimination. A direct muscle stimulation with a pattern said to mimic the activity pattern of “fast” motor neurons accelerates, by 2–3 days, the developmental synapse elimination in rat SOL76. Early studies suggest selective innervation of different fiber types, each by motor neurons with an appropriate activity pattern79. Other studies, however, demonstrated a significant degree of mismatches during early postnatal development81 that would be expected following pervasive innervation of target muscles during early development7. Such findings suggest that refinement of neuromuscular connections during postnatal maturation would necessitate preferential pruning of inputs with inappropriate activity patterns. Both scenarios are compatible with the current observation where the resolution of synapse elimination on distinct fiber types is temporally offset. Interestingly, a recent study indicates that “fast/slow identity” of postsynaptic muscle fibers can influence that of the innervating motor neurons80. This may imply that any significant changes to the sizes of motor units (both fast and slow) at the peak of polyneuronal innervation – which could indirectly alter the timing of synapse elimination – need not occur. This observation, on the other hand, suggests that it is just as possible for muscle fibers to influence synapse elimination indirectly via alterations of partnering motor axons as it is to do so directly.A motor neuron and the set of muscle fibers it innervates compose a motor unit. Within a mature mammalian motor unit, all the muscle fibers share a common fiber type82, may actively repel immature axons of “fast” motor neurons. Class 1 major histocompatibility complex (MHC1) molecules, whose genetic inactivation leads to a delay in synapse elimination, both at the NMJs70 as well as the relay cells of the visual system within the lateral geniculate nucleus83, also provide a family of attractive candidates that may be involved in recognition and removal of non-compatible synaptic partners. Protracted synapse elimination found in neural cell adhesion molecule (NCAM) deficient animals, however, appears to stem indirectly from altered presynaptic activity57. On the other hand, the reported upregulation of NCAM by denervated muscle fibers84 may be interpreted as an attempt to provide a more permissive substrate for any axons that may subsequently come to innervate them regardless of the downstream mechanism. Secreted factors – such as brain-derived neurotrophic factor (BDNF), whose expression and functional maturation is regulated by activity85 or fibroblast growth factor binding protein 1 (FGFBP1)86 – present another potential avenue through which distinct muscle fibers may influence local synaptic competition. It is presently unknown whether distinct muscle fiber types differentially express BDNF, FGFBP1, MHC1 or NCAM. Additionally, presumed differences in activity levels of motor neurons that innervate slow and fast fibers may lead to differential expression of type-III neuregulin187, a motor neuron-derived factor that influences the timing of synapse elimination through the activation of tSCs20.The mechanism(s) by which appropriate matching of pre- and postsynaptic partners is achieved remains obscure. Even if the developmental scenario requires that motor neurons seek out appropriate target fiber types for initial innervation during initial synapse formation, such ability for the motor axon and/or the target muscle fiber to recognize the correct synaptic partner would be necessary. Whether direct or indirect, muscle fibers must be able to discriminate between appropriate and inappropriate motor inputs or provide the motor neurons the means to do so. A repulsive axon guidance molecule ephrin-A3, recently found to be expressed specifically in slow fibers postnatally95 may suggest preferential denervation of fast fibers that transdifferentiate into slow fibers upon re-innervation by a slow motor axon. Several recent studies of amyotrophic lateral sclerosis (ALS)101, the disease etiology of which appears not to be motoneuron-autonomous103, provide additional support for this idea. NMJs of slow fiber-rich muscles – SOL and TS – display a delayed degeneration compared to fast fiber-rich muscles105. The relative sparing of these slow muscles to disease progression likely stems from the reported resistance to denervation of NMJs situated on slow fibers101. These findings may further suggest that, at least in muscles with significant slow motor neuron innervation, the two distinct pools of motor neurons – one compensatory pool that grows sprouts to occupy the endplates left vacant by the dying-back axons of the other pool104 – may partition with the contractile properties of their target muscle fibers. The observation that the integrity of NMJs found on slow fibers is relatively refractory to neuromuscular pathology appears not to be isolated to ALS. Muscle-specific overexpression of PGC1α, and therefore an increase in slow fibers, is also reported to alleviate the decline in the integrity of NMJs associated with sarcopenia106 and a mouse model of muscular dystrophy107. The present findings, in which the timing of synapse elimination was altered through muscle fiber-specific genetic manipulation, motivate a search for muscle-derived factors differentially expressed among fiber types that confer increased stability to neuromuscular synaptic connections. Such synapse stabilizing factors potentially represent a novel means to treat ALS and other neurodegenerative diseases with synaptic degeneration early in disease progression. Consistent with such therapeutic potential of synapse stabilizing factors, enhanced activation of muscle-specific kinase – necessary for both formation and maintenance of NMJs110 – either by genetic or pharmacological means – delays ALS disease progression in a mouse model112.In light of the present results, it is tempting also to speculate on whether the motor endplates of slow fibers are less vulnerable, compared to fast fibers, to age- and disease-induced denervations. Evidence of both a rearrangement of the distribution of muscle fibers within motor units as well as a prominent shift in the muscle contractile properties towards a slower, more fatigue-resistant characteristic in aged rodents and humansSUPPLEMENTARY INFO


The secondary flow generates uneven sedimentation and adherence that negatively impacts cell culture quality. Here we show a modification of the circular culture vessel that abrogates these disturbances. Cell culture wells were augmented with a central column to diminish secondary flow. Human carcinoma cell lines , mesenchymal stem cells [human umbilical cord perivascular cells (HUCPVC)] and mouse embryonic fibroblasts (MEF) were cultured in both column-augmented and regular culture wells. Human carcinoma cell cultures showed even cell densities and significantly more viable cells in column-augmented vessels. In FTM HUCPVC cultures, cell surface MSC marker  expression and cell differentiation-related gene expression patterns were significantly more homogeneous in column-augmented vessels. MEF cells in column-augmented culture vessels showed a more consistent expression of IGF-1. Column-augmented cell culture vessels significantly improve the homogeneity of adherent cell cultures by mitigating the adverse effect of the secondary flow.High quality cell cultures require reliable laboratory practices. Today's small-scale This article has an associated First Person interview with the first author of the paper. Summary: Secondary flow in small scale cultures can crucially affect cell phenotype and function. A simple modification of the classical round-shaped cell culture vessels can minimize the adverse effect of secondary flow. The principles of small-scale cultures date back to Julius Richard Petri's research in the 1880s . In any academic or industrial establishment, maintenance and investigation of mammalian cell cultures begins with the traditional low-volume plasticware, typically made of polystyrene (PS) or polyethylene (PE). The most commonly used are 10 cm and 6 cm diameter culture dishes. Six- to 96-well plates provide a sufficient surface for 103-106 adherent cells to develop, ideally as monolayer cultures. Cell densities in these cultures are set to suit the purpose of the culture and typically range between 20–70% surface coverage, and this is referred to as ‘confluency’. Procedures and assessments requiring limited cell–cell connections and high relative cell surface (such as transfection protocols) or identifiable cell margins (microscopy) favor lower cell confluency, while tests conducted on larger cell populations (flow cytometry and immunoblotting) seek higher yields from a single vessel. Regardless of preferred confluence, consistent growth of the culture is key to ensure any treatments to the cell population are evenly applied and the subsequent results sufficiently represent the entirety of the cells in the culture.In addition to the practical aspects of the analysis, the experimental design must consider that the cell density in a culture fundamentally impacts the micromilieu and as such, the biology of the cells. Higher local cell densities increase autocrine and paracrine effects , and celIn order to have sufficient control over cell cultures and guarantee stability and reproducibility, physical parameters are pre-set and monitored constantly. These parameters include temperature, oxygen and carbon dioxide levels, and, if possible, the culture media composition. The control over the culture's cell density is just as crucial because cells are essential environmental factors for each other. Setting defined cell numbers at seeding and performing counting at the time of harvest accounts for the overall cell numbers per culture unit. However, local alterations in cell confluency and cell growth can lead to inhomogeneities in the cell culture and result in variability within the cultivated cell population. Uneven seeding, adhesion and proliferation can lead to these heterogeneities that, depending on the cell type, can give rise to uncontrolled, phenotypically diverse sub-populations. Cell culture protocols are stringent practices used in an attempt to homogenize cell populations and eliminate factors that might jeopardize even growth and homogeneity.The so called ‘tea-leaf paradox’ was known for centuries and posed a non-trivial fluid mechanics puzzle until 1926, when Albert Einstein published his article on the matter . Tea leaSecondary flow occurs in any fluid phase moving along a curved path. It is most prominent in cylindrical vessels where fluids are subject to forces that set the liquid in circular motion. Dispersed particles in the liquid move with the secondary flow and sediment along the smaller curve or the center of a cylindrical vessel.Research Gate). Although other effects of the fluid flow on cells such as shear stress  secondary flow is induced when handling cylindrical culture vessels, leading to uneven attachment and growth of adherent cells; and (2) placing a circular object in the center of the culture vessel prior to seeding will decrease secondary flow and increase homogeneity in an adherent cell culture.Mesenchymal stromal cells or mesenchymal stem cells (MSC) are receiving increased attention and attracting funding in the field of regenerative medicine. Since the introduction of the concept of adult stem cells  and the We are investigating a novel young source of MSC isolated from the perivascular region of the human umbilical cord . These pWe tested our hypothesis by utilizing six-well tissue culture plates (35 mm diameter wells) and straight, cylindrical polystyrene inserts attached to the center of flat bottom wells with biologically inert silicon grease. Assigned wells were augmented with 7 mm diameter central attachments, henceforth referred to as ‘columns’. Consequently, the ratio of the inner and outer radii of the attachment surface was 1:5 in column-augmented wells C. The coMovie 1). In regular, unaltered wells sedimentation inhomogeneities were observed instantly with a high density of beads appearing in the wells' central area (Movie 1). However, in column-augmented wells (Movie 1) latex beads showed even sedimentation and distribution throughout the wells.For rapid visualization of liquid flow and sedimentation in regular and column-augmented wells, latex beads (4 µm) were transferred into each well of the six-well plates as a 1:5 dilution with PBS (3 ml). Beads were noted to visibly begin to sediment after the tissue culture plate was randomly agitated on the horizontal plane  and cultured for 3 days. At the time of seeding, cell culture plates were subjected to the movements occurring in our usual laboratory practices, with no additional shaking or agitation applied to the culture plates. 3 days after seeding, cultures were imaged by phase contrast microscopy . In each six-well plate three wells were unaltered and three were column-augmented. With all wells physically connected, any force applied on the plate affected all wells identically. To mimic the manipulation of the culture plate and the conventional mixing practices that aim to distribute cells evenly in culture plates, we moved the plate 3–4 times rapidly along the horizontal plane before placing it into the incubator. After 48 h of incubation  phase contrast and fluorescence microscopy were performed to assess cell growth and confluency  . SamplesMSC are regularly detected and characterized by specific cell surface markers that can individually or in combination identify MSC populations, distinguishing them from other cell types and indicate their undifferentiated state . CD105 . While CChallenging the paradigm of the constitutive representation of MSC-markers, research shows that CD105 expression can be controlled by oxygen availability in the cellular micromilieu , while C4 cells were incubated with either CD105 (PE) or CD90 (APC) antibodies and analyzed using MACSQuant digital flow cytometry. Mean fluorescence intensities (MFI) were exported and graphed to compare signals contributed to cells grown in the central or peripheral regions of each culture well , pluripotent (PS) and neural (NS) stem cells and whose expression level has become a quality control measure when comparing feeder cells . Cell culture plates were handled according to standard laboratory practices and included plate transfer for bright field microscopy from the biosafety cabinet to a humidified incubator. No additional agitation was applied to the plates. After 3 days, MEF cultures from regular wells showed similar differences in cell densities that we observed for the cell types discussed previously  we demonstrated that inhomogeneities in the growth of these adherent cell cultures can occur during routine growth in conventional, cylindrical culture vessels. Finally, we tested MEF to assess whether their function of generating supportive niche for stem cells is impacted by the inhomogeneities in the typical culture system. Our experimental results were consistent for each cell type tested: the differences in confluency found concurrently in the same culture vessel can lead to alterations in the cells' immunophenotype and gene expression regulation and can ultimately lead to the emergence of phenotypically different sub-populations. Therefore, gaining control over the homogeneity of cell cultures ensures the reliability of the evaluation and increases the fidelity of the transition towards large-scale applications.Cultures of immortalized cell lines are common tools for basic science and research given their general robustness and ease of handling. However, we found that inhomogeneities emerging in BeWo and JEG-3 cultures cultivated in circular vessels negatively impact their growth and viability. Highly proliferative cells suitable for culturing and We have demonstrated here, with a simple modification, that augmenting standard circular culture vessels with a central insert, or ‘column’, growth homogeneity of the adherent culture can be significantly increased, and the adverse effects of uneven cell growth can be virtually eliminated. Based on these findings, we suggest that such an approach should be strongly considered when working with adherent cell cultures.Six-well cell culture plates were purchased from Corning. Sterile polypropylene inserts of 35 mm in diameter were applied as columns attached to the bottom surface of selected culture wells using sterile silicon grease (Eppendorf).4 µm silicone beads were used for cell-free sedimentation assessment (Thermo Fisher Scientific). Silicone beads were washed with PBS and transferred into culture wells in a 1:5 dilution in 3 ml of PBS. Sedimentation was visualized after rapid agitation.4 cells were seeded in 2 ml media per well in six-well cell culture plates. Cells were analyzed 3 days after seeding.JEG-3 and BeWo human choriocarcinoma cell lines were ordered from ATCC and cultured in alpha MEM (Gibco) supplemented with 10% fetal bovine serum  and 1% Penicillin-Streptomycin (Gibco). For cell growth and viability assessments, 2.5×104 cells in 3 ml of media (as described above). Rapid horizontal agitation was applied to culture plates containing both regular and column-augmented wells. Cell cultures were incubated for 48 h at 37°C, 5% CO2, in humidified incubators. Bright field (Zeiss) and fluorescence microscopy  images were acquired after 48 h of incubation. Images were taken of central and peripheral fields of both regular and column-augmented wells.Three lines of FTM HUCPVC (passage 4–6) were cultured in alpha MEM (Gibco) supplemented with 10% fetal bovine serum  and 1% Penicillin-Streptomycin (Gibco). FTM HUCPVC were transferred into culture wells as single cell suspensions of 5×105 cells were prepared and incubated with primary conjugated antibodies (1:40 dilution in PBS with 3% FBS). Digital flow cytometry was performed using MACSQuant (Miltenyi). Data analysis was performed with FlowJo software. Statistical analysis was performed using ANOVA.Cell Tracker Green (Invitrogen) life fluorescent dye was applied to adherent cell cultures at 2.5 µg/ml for 30 min to load cells for fluorescence microscopy and well-scan. Quantitative fluorescence well scans were performed on fluorophore-labelled cell cultures using a digital plate reader . Immunofluorescence labelling of FTM HUCPVC was performed using primary conjugated antibodies: anti- CD105 PE (RnD) and anti-CD90 APC (BD). Antibodies were applied in a 1:40 dilution in PBS supplemented with 3% FBS (Hyclone) for 30 min. Fluorescence imaging of immuno-labelled cells was performed with an EVOS digital microscope (Life Technologies). For flow cytometry analysis single cell suspensions of 105 cells per sample for qPCR analysis. Cells were lysed and RNA was purified using the RNEasy Plus Mini Kit according to the manufacturer's instructions. Quantitative PCR was performed using the Human Cell Lineage Identification PCR Array  run in a Corbett RotorGene qPCR machine (Qiagen). Gene expression levels were normalized to integrated housekeeping controls . Results were analyzed and exported by the RT2 Profiler PCR Array Data Analysis Platform . Results were expressed as a fold-change between samples gathered from the peripheral and central areas of the culture wells.RNA was isolated from 102 in a humidified incubator. After 72 h in culture, cells were lysed from the central and peripheral growth surfaces separately using RLT buffer (Qiagen). RNA was purified using the RNeasy Mini Kit (Qiagen). cDNA was synthesized with First Strand RT2 (Qiagen) and qPCR was performed using Quant Studio5 (Thermo Fisher Scientific) using SYBR Green Master Mix (Qiagen). Gene expression levels were normalized to housekeeping control β-actin; primers were synthesized by ACGT Corp. (Toronto). Mouse IGF-1 Forward: AAAGCAGC-CCCGCTCTATCC, Reverse: CTTCTGAGTCTTGGGCATGTCA, Mouse β-actin Forward: GGCACCACACCTTCTACAATG, Reverse: GGGGTGTTGAAGGTCTCAAAC. Results were analyzed with the RT2 Profiler PCR Array Data Analysis Platform . The thermal profile for qPCR was 95°C for 10 min followed by 40 cycles of 15 s at 95°C, with 2 min at 65°C. Relative expression of the target genes was calculated with the 2¯ΔΔct method.Mouse Embryonic Fibroblast Feeder cells (Applied Stem Cell Inc.) were cultured in DMEM (Sigma Life Science) supplemented with 10% FBS (Hyclone), 1% Penicillin-Streptomycin (Gibco), and 1 mM Sodium Pyruvate (Gibco). Cell cultures were maintained at 37°C, 5% CO


Heligmosomoides polygyrus to test the hypothesis that pathology specifically within the small intestine caused by a helminth co-infection would influence oral prion disease pathogenesis. When mice were co-infected with prions on d 8 after H. polygyrus infection the early accumulation of prions within Peyer’s patches was reduced and survival times significantly extended. Natural prion susceptible hosts such as sheep, deer and cattle are regularly exposed to gastrointestinal helminth parasites. Our data suggest that co-infections with small intestine-restricted helminth pathogens may be important factors that influence oral prion disease pathogenesis.The early replication of some orally-acquired prion strains upon stromal-derived follicular dendritic cells (FDC) within the small intestinal Peyer’s patches is essential to establish host infection, and for the disease to efficiently spread to the brain. Factors that influence the early accumulation of prions in Peyer’s patches can directly influence disease pathogenesis. The host’s immune response to a gastrointestinal helminth infection can alter susceptibility to co-infection with certain pathogenic bacteria and viruses. Here we used the natural mouse small intestine-restricted helminth pathogen  Sc, abnormally folded isoforms of the host-encoded cellular prion protein (PrPC), accumulate in the affected tissues of prion disease infected hosts and are considered to constitute the major component of the infectious agent3. Many prion strains such as natural sheep scrapie, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in cervid species can be orally acquired. Furthermore, some prion strains can have zoonotic potential as the consumption of BSE-contaminated food was responsible for the subsequent emergence of variant Creutzfeldt-Jakob disease (vCJD) in the UK human population4. Data from experimental mice5, sheep with natural scrapie6, deer with CWD7 and vCJD patients8 show that some orally acquired prion strains replicate first upon stromal-derived follicular dendritic cells (FDC) in the gut-associated lymphoid tissues (GALT) as they make their journey from the gut to the brain (termed neuroinvasion). Mouse studies show that the initial replication of the prions within the GALT is essential to establish host infection12. Prions then spread from the GALT via the enteric and peripheral nervous systems to the brain where there accumulation ultimately leads to neurodegeneration and death14.Prion infections cause chronic neurodegenerative diseases in humans and animals for which there are no treatments. Aggregations of PrPPRNP genotype (which encodes PrPC) and prion agent strain can also influence an individual’s susceptibility to oral prion infection. For example, the adverse effects of ageing on the GALT microarchitecture impede the early uptake and replication of orally-acquired prions within these tissues and reduce disease susceptibility16. This may help to explain why the majority of the clinical vCJD cases have been predominantly recorded in young individuals 18.The incidence of clinical cases of vCJD in humans has fortunately been much lower than originally estimated despite the probable widespread exposure of the UK population to BSE-contaminated food in the 1980s. This implies that factors in addition to host 21. Oral prion disease is blocked in mice lacking these cells22, and exacerbated in mice with an increased M cell-density22. Oral infection with certain pathogenic bacteria or exposure to inflammatory stimuli such as cholera toxin can increase M-cell density in the intestine25, and with the damage and/or inflammation caused by pathogen infection26 this could potentially exacerbate oral prion disease pathogenesis by increasing the efficiency of the initial uptake of the prions from the gut lumen. Mononuclear phagocytes (MNP) are a diverse population of macrophages and classical dendritic cells (DC)29 that also play important and contrasting roles during prion disease depending on the subset30. Whereas CD11c+ classical DC aid the propagation of orally acquired prions towards FDC in Peyer’s patches in order to establish infection32, the uptake of prions by macrophages can lead to their destruction34. Therefore, pathogen-induced alterations to the abundance, trafficking or activity of MNP could similarly influence disease pathogenesis by enhancing the propagation or clearance of orally-acquired prions.Pathogen induced alterations to GALT also have the potential to influence prion uptake and disease susceptibility. For example, orally-acquired prions are initially transported across the gut lumen into Peyer’s patches by a specialized population of phagocytic epithelial cells known as M cells35 including Salmonella enterica serovar Typhimurium36, Citrobacter rodentium37 and norovirus38. Natural prion susceptible hosts such as sheep, deer and cattle are regularly exposed to gastrointestinal helminth parasites, but little is known of the effects that co-infection with these pathogens may have on oral prion disease pathogenesis. Since neuroinvasion of orally-acquired prions occurs directly from the GALT in the small intestine12, in this study the natural mouse small intestine-restricted helminth pathogen Heligmosomoides polygyrus was selected. This parasite is an excellent model for the study of gastrointestinal helminth infections in livestock and humans39, being phylogenetically similar to the ruminant parasites Haemonchus contortus and Teladorsagia circumcincta, and human hookworms Ancylostoma duodenale and Necator americanus40. We used H. polygyrus to test the hypothesis that the pathology caused by a pathogen co-infection specifically within the small intestine would significantly influence oral prion disease pathogenesis. These data are essential for the identification of important factors can that influence the risk of oral prion disease transmission and to help design effective intervention and control strategies.Studies in mice have shown that the host’s immune response to a gastrointestinal helminth infection alters susceptibility to co-infection with a variety of other pathogenic microorganismsH. polygyrus L3 larvae by gavage and faecal egg burdens measured at intervals afterwards to monitor the magnitude of the parasite infection. As anticipated, maximum egg production was observed by 18 days post-infection (dpi) with H. polygyrus and had declined by 32 dpi  confirmed the presence of detectable pathology within the small intestine by 8 dpi with H. polygyrus, and was significantly greater in the duodenum by 14 dpi  analysis showed that these structures typically comprised a central core which predominantly contained CD11b+ MNP, surrounded by other populations of CD68+ and CD11c+ MNP  were orally-infected with H. polygyrus and 0, 1, 8 or 14 d later the mice were subsequently orally co-infected with a limiting dose of ME7 scrapie prions. The days on which the mice were co-infected with prions were selected based on the known pathogenesis of the H. polygyrus infection in the small intestine44: 1 dpi, when the infective L3 larvae penetrate the lining of the duodenum; 8 dpi, during the period when the adult worms re-emerge back into the gut lumen; 14 dpi, when adult worms are tethered around the villi in the duodenum. A separate group of mice were orally-infected with prions alone as a control.Next, groups of C57BL/6J mice  that are associated with a terminal, clinical, infection with ME7 scrapie prions . The distribution and severity of the spongiform pathology was also similar in the brains of the mice that developed clinical signs of prion disease , microgliosis, astrogliosis and accumulations of prion disease-specific PrP  PrPSc accumulations were first detected in a small percentage of the Peyer’s patches by 35 dpi and had increased in frequency by 105 dpi  that covers the Peyer’s patches can directly influence oral prion disease susceptibility22. Whole-mount IHC showed that the density of M cells expressing the mature M-cell marker glycoprotein 2  in FAE covering the Peyer’s patches was unchanged after H. polygyrus infection  were orally infected with H. polygyrus and at 5 dpi orally-gavaged with fluorescent microbeads. Uninfected mice were orally-administered fluorescent microbeads as a control. Seventy two hours later (d 8 post-H. polygyrus infection) the number of microbeads that had been transcytosed into the subepithelial dome (SED) region of their Peyer’s patches was quantified on tissue sections by fluorescence microscopy. Similar levels of fluorescent microbeads were detected in the SED regions of the Peyer’s patches from mice from each group  manner, and CR1 (CD35) is considered to play a prominent roleoup Fig. . The oveion Fig. . In contice Fig. . These d+ MNP to mediate their delivery to FDC32, whereas macrophages appear to phagocytose and destroy prions34. IHC and morphometric analysis suggested that the % area of CD11c+  in the Peyer’s patches was unchanged during H. polygyrus infection  2 immune responses associated with expression of cytokines such as interleukin (IL)-4 and IL-13, and potent regulatory T-cell responses54. However, data show that deficiency in T cells58 or the cytokines59 IL-4 or IL-13 do not influence peripheral prion disease pathogenesis or susceptibility. This suggests that the effects of helminth co-infection on oral prion disease pathogenesis were similarly independent of effects on T cells.Infections with gastrointestinal helminth parasites such as + MNP such as the classical DC in the underlying SED regions of the Peyer’s patches51. The propagation of prions by these MNP towards the FDC in the Peyer’s patches is important to establish host infection and efficient neuroinvasion32. Our analysis revealed that the abundance of CD11c+ MNP in the B cell mantle region of the Peyer’s patches was specifically and significantly increased on d 8 after H. polygyrus infection. This implied that the disturbed distribution of the MNP within the Peyer’s patches on d 8 after H. polygyrus infection may have impeded the ability of these cells to efficiently propagate the prions from the SED region down towards the FDC within the B cell follicles. Other MNP populations such as macrophages appear to play a host protective role during prion disease by aiding the clearance of prions34. The “alternative” activation of macrophages is a characteristic feature of the helminth-induced Th2 response60. These alternatively-activated macrophages typically contribute to the regulation of inflammatory responses, and promote wound healing and parasite expulsion during helminth infections39. Whether effects of H. polygyrus infection on the activation status of the MNP in the Peyer’s patches may have also enhanced their ability to phagocytose and destroy the prions remains to be determined.Following their initial transcytosis across the gut epithelium by M cells the prions are subsequently acquired by CD11cH. polygyrus infection that were responsible for the altered positioning and trafficking of the CD11c+ MNP within the Peyer’s patches are not known. Day 8 after H. polygyrus infection was selected as it is around this time that the adult worms emerge from the submucosa into the lumen of the duodenum44. During this process, significant amounts of parasite-derived antigens and debris from damaged host cells are liberated into the gut lumen. Thus, it is plausible that the presence of this material in the gut lumen stimulates the recruitment of CD11c+ MNP to the Peyer’s patches.The factor/s during 32. In mice co-infected with prions on d 8 after H. polygyrus infection the accumulation of prions within the MLN was also reduced despite the presence of larger FDC networks. This suggests that any prions that are initially transported to the MLN immediately after oral exposure either reach this tissue in insufficient abundance to establish infection on FDC, or alternatively, are not propagated towards the B cell follicles. As a consequence, the prions are most likely phagocytosed and destroyed by the macrophages within the MLN34.Orally-acquired prions accumulate first in the small intestinal Peyer’s patches and subsequently spread via the lymphatics and bloodstream to most other secondary lymphoid tissues, including the MLN and the spleen. However, our previous data show that the subsequent accumulation of the prions within these tissues is not essential for efficient neuroinvasion from the intestine64, consistent with data from experimental oral transmissions to mice12. Natural prion disease susceptible hosts such as sheep, deer and cattle are regularly exposed to helminths in field situations. Thus, co-infection with small intestine-restricted helminth pathogens around the time of oral exposure may similarly influence oral prion disease pathogenesis in naturally affected host species. An independent study has previously attempted to determine the influence that helminth infections may have on oral prion disease pathogenesis65. The gastrointestinal nematode parasite T. circumcincta establishes infection in the mucosa and lumen of the abomasum of sheep. When lambs with high genetic susceptibility to natural sheep scrapie were experimentally co-infected with T. circumcincta at monthly intervals from 6 to 11 months old, prion disease survival times were shortened65. However, very young lambs are highly susceptible to prion infection66 and are considered to acquire natural sheep scrapie from infected ewes via the oral route around the time of birth67. Treatments that can delay the accumulation of prions in the GALT do not influence disease pathogenesis once neuroinvasion has occurred and the prions have established infection in the peripheral nervous system5. Since the lambs in the above study65 were co-infected with T. circumcincta long after prion neuroinvasion from the intestine is likely to have occurred62, the reduced survival times are unlikely to be a consequence of effects of the parasite infection on the uptake or accumulation of the prions within the GALT. However, it is plausible that the parasite-mediated damage to the abomasum stimulated the systemic release of inflammatory mediators that exacerbated the development of neuropathology within the CNS68.Studies of sheep with natural scrapie have revealed that orally acquired prions are similarly first detected in the GALT in the upper gastrointestinal tract before spreading to the draining lymph nodes and onwards to other lymphoid tissues. The large intestinal GALT such as the caecal patches are not early sites of prion accumulationH. polygyrus. Although experimental helminth infections in mice are commonly studied after administration of a single dose, natural host species in field situations are likely to be regularly exposed to these pathogens over long periods. The effects that chronic helminth exposure may have on MNP abundance and distribution within the Peyer’s patches are not known. It is plausible that repeated helminth exposure may lead to the prolonged recruitment of MNP into the B cell mantel region of the Peyer’s patches further enhancing the sequestration of orally-acquired prions.In the current study mice were given a single infection with C-expressing stromal cell population in experimentally-induced soft tissue granulomas can support prion replication in infected mice43. Similar evidence of ectopic PrPSc accumulation has been observed in the newly formed lymphoid follicles of chronic lymphofollicular inflammatory sites in scrapie-affected sheep69. Granulomas can form around persistent foreign bodies, and are induced in the intestines of H. polygyrus infected mice in response to the invasion of the L3 larvae into the submucosa. However, the granulomas induced in the intestines of H. polygyrus infected mice predominantly contained MNP, lacked organized lymphoid follicles and PrPC-expressing stromal cells within them. Although the granulomas appeared to be surrounded by a border of PrPC-expressing stromal-like cells they were unable to support PrPSc accumulation in mice co-infected with prions.Soft tissue granulomas are a prominent feature within chronically-inflamed tissues in humans and animals. One study has shown that an inflammatory PrP26. However, our own studies using two distinct gastrointestinal helminth pathogens show that the damage these parasites cause to the mucosa in the small (current study) and large intestines12 has little, if any, effect. Instead our data suggest that oral prion disease pathogenesis is significantly altered when the density or the status of the cells utilized by prions to establish infection within small intestinal Peyer’s patches is affected, including: M cells22; MNP34 (current study); and FDC16. Natural prion disease susceptible hosts such as sheep, deer and cattle are regularly exposed to helminths. Thus, co-infection with small intestine-restricted helminth pathogens around the time of oral prion exposure may be important factors that can influence prion disease pathogenesis in naturally affected host species.Minor oral  lesions can enhance susceptibility to CWD prions in cervid PrP-expressing transgenic mice by facilitating direct neural invasionApprovals for the mouse studies and regulatory licences were received from The Roslin Institute’s and University of Edinburgh’s ethics committees, and the UK Home Office. The animal experiments described in this study were undertaken under the authority of UK Home Office Project Licences (PPL60/4325 & PA75389E7) under the terms, conditions and regulations of the UK Home Office ‘Animals (scientific procedures) Act 1986’. Where appropriate and required, necessary efforts were undertaken to reduce harm and suffering. Mice were humanely culled at the end of the experiments using a UK Home Office Schedule One method.Six-eight weeks old female C57BL/6J  were used throughout this study. All mice were maintained under SPF conditions.H. polygyrus third stage (L3) larvae prepared as described70. Egg counts were determined by weighing faecal pellets from individual mice prior to resuspension in 1 ml of saturated sodium chloride/50% w/v glucose solution. Eggs were then counted using a McMaster chamber and the eggs/g faeces determined. For analysis of pathology, blinded sections of duodenum and ileum (2 per mouse) were scored for inflammatory cell infiltrate (0–4) and changes to the intestinal architecture (0–4) using the evaluation scheme described for H. polygyrus pathology in41. Mean scores per mouse were combined to give a maximum score of 8.Mice were orally infected by gavage with 200 10 intracerebral ID50 units. Each mouse was then individually housed in a bedding- and food-free cage with water provided ad libitum, and fed an individual prion doused food pellet31. The mice were returned to their original cages as soon as no signs of the food pellet were evident (indicating that it had been completely ingested). The mice were then coded and assessed by independent technicians at weekly intervals for the clinical signs of prion disease and scored as “unaffected”, “possibly affected” and “definitely affected” using standard criteria established for mice clinically-affected with ME7 scrapie prions31. Mice were either culled at the standard clinical endpoint, or survival times recorded for those that remained free of the clinical signs of prion disease after exposure. The clinical status in each mouse was confirmed by the histopathological assessment of prion disease pathology in the brain. The magnitude and distribution of the spongiform pathology (vacuolation) in nine distinct grey-matter regions of the brain was compared as described71.Individual food pellets were first doused with 50 µl of scrapie brain homogenate derived from mice with terminal ME7 scrapie prion infection. Each pellet contained approximately 3.3 log21 Peyer’s patches were first fixed using BD Cytofix/Cytoperm , and then immunostained with rat anti-mouse GP2 mAb . The Peyer’s patches were then stained with Alexa Fluor 488-conjugated anti-rat IgG Ab , rhodamine-conjugated Ulex europaeus agglutinin I  and Alexa Fluor 647-conjugated phalloidin to detect F-actin (Invitrogen).To detect M cells by whole-mount immunostaining72) to detect cellular PrPC. To detect MNP, sections were immunostained with hamster anti-mouse CD11c mAb , rat anti-mouse CD11b mAb  or rat anti-mouse CD68 mAb . Rabbit anti-synaptophysin 1  was used to detect nerve synapses. Sections were subsequently immunostained with streptavidin-conjugated or species-specific secondary antibodies coupled to Alexa Fluor 488 (green), Alexa Fluor 594 (red) or Alexa Fluor 647 (blue) dyes (Life Technologies). Cell nuclei were detected using DAPI . Sections were mounted in fluorescent mounting medium (DAKO) prior to imaging on a Zeiss LSM710 confocal microscope .Intestines and MLN were snap-frozen at the temperature of liquid nitrogen, and 6 µm serial frozen sections cut using a cryostat. To detect FDC, sections were immunostained with anti-mouse CD35 mAb to detect CR1  and PrP-specific polyclonal antibody (pAb) 1B3 (ref.Goblet cells were detected using a Periodic Acid Schiff (PAS) Stain Kit  according to the manufacturer’s instructions.d) in intestines, MLN and brains were first fixed in periodate-lysine-paraformaldehyde fixative and then embedded in paraffin wax and 6 µm sections cut. The sections were first pre-treated with a combination of hydrated autoclaving  followed by 5 min immersion in formic acid (98%) to enhance PrPd detection, before immunostaining with 1B3 PrP-specific pAb. Astrocytes were detected by immunostaining with anti-glial fibrillary acidic protein . Microglia were detect by pre-treatment with citrate buffer, followed by immunostaining with anti-ionized calcium-binding adaptor molecule 1 . To detect FDC, the sections were pre-treated with Target Retrieval Solution (DAKO) before immunostaining with rat anti-CD21/35 mAb . Sections were counterstained with biotin-conjugated species-specific secondary antibodies  followed by HRP-conjugated to the avidin-biotin complex  with DAB (Sigma) as a substrate. Haematoxylin was used as a counterstain to detect cell nuclei. Paraffin-embedded tisssue immunoblot analysis73 was used to detect proteinase K-resistant PrPSc using with PrP-specific pAb 1B3.To detect disease-specific PrP (PrPhttp://rsb.info.nih.gov/ij/) was used for morphometric image analysis on coded sections as described74. First, pixel intensities across all the areas of each image (immunostained and non-stained regions) were measured using an ImageJ macro and used to obtain the background pixel intensity threshold values. These values were then applied to all subsequent analyses. The number of pixels in each colour channel  in each image were then determined automatically. These data are presented as the proportion of positively immunostained pixels/total number of pixels  in the specific region of the image. We typically analysed 1–7 images from each mouse, and multiple mice were analysed from each group (n = 3–8 mice/group). The figure legends display the sample sizes for every parameter analysed by this method in this study. For analysis of CD11c+ cell location, images were orientated with the FAE at facing upwards and the height of the patch and the Y-axis coordinate of each CD11c+ cell recorded. The location of each CD11c+ cell was calculated as a percentage of the total patch height and then allocated to three regions approximately representing the B cell follicle , B cell mantle (33.34–66.67%) and SED (66.68–100%).ImageJ software  in 200 µl PBS. Mice were culled 72 h later and Peyer’s patches and small intestine segments were snap-frozen at the temperature of liquid nitrogen. Serial frozen sections (6 µm in thickness) were cut on a cryostat and counterstained with DAPI. The number of beads in the SED from 3 sections of two Peyer’s Patches per mouse  were counted. Images of SED were acquired using Nikon Eclipse E400 fluorescent microscope using Micro Manager (http://www.micro-manager.org). Tissue auto-fluorescence was subtracted from displayed images using ImageJ.Mice were given a single oral gavage of 2 × 10P < 0.05 were accepted as significant.All data are presented as mean ± SD. Unless indicated otherwise, differences between groups were analysed by One- or Two-way ANOVA where appropriate. Comparisons between groups were performed using a Tukey’s (One-way ANOVA) or Sidak’s (Two-way ANOVA) post-hoc test. In instances where there was evidence of non-normality , data were analysed by appropriate non-parametric tests. Values of


African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a re-expanding devastating and highly lethal hemorrhagic viral disease. microRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. The discovery of virus specific miRNAs has increased both in number and importance in the past few years. We have recently described the differential expression of several porcine miRNAs during in vivo infection with attenuated and virulent ASFV strains. Here, we have extended these studies trying to identify the presence of viral miRNAs encoded by ASFV in an in vivo infection in pigs.Sixteen small RNA libraries were analyzed from spleen and submandibular lymph nodes obtained from eight pigs, seven infected with either the virulent E75 ASFV strain or its attenuated counterpart E75CV1, or from pigs surviving E75CV1-infection and challenged with BA71  and one non infected as negative control. Samples were recovered at different times post-infection. Libraries were analyzed by next-generation sequencing. Some viral miRNA candidates were initially identified, which did not correspond to porcine miRNAs. Further structural analyses were carried out in order to confirm if they met the conformational requirements to be considered a viral miRNA.The analysis of sixteen small RNA libraries prepared from two different tissues obtained from pigs experimentally infected with E75, E75CV1 or with E75CV1 plus BA71, revealed the presence of six potential miRNA sequences but none of them met the requirements to be considered as viral miRNAs. Thus, we can conclude that ASFV does not express miRNAs in vivo, at least under the experimental conditions described here. Asfarviridae family, genus Asfivirus. ASFV is a large icosahedral, enveloped, double stranded (ds) DNA virus ranging from 170 to 193 kb [. Lack of vaccines or treatments complicates its control [Herpesviridae, Polyomaviridae, Adenoviridae, Papillomaviridae, Baculoviridae, Ascoviridae and Iridoviridae [Ascovirus and Iridovirus, together with Asfivirus, belong to the monophyletic superfamily of nucleocytoplasmic large DNA viruses (NCLDV). Thus, ASFV was a good candidate for a virus capable of encoding miRNAs. On the other hand, recently, our group has identified differential expression of porcine miRNAs in pigs after the infection with ASFV [African swine fever (ASF) is a contagious disease that affects domestic and wild pigs caused by ASF virus (ASFV) belonging to the o 193 kb . Lack of control . The vir control . The stu control . In addioviridae –10. All ith ASFV .Considering this background, the aim of the present study was to explore if ASFV can encode viral miRNAs in an experimental infection in its natural host by using high-throughput sequencing. To this end, in order to characterize the potential presence of ASFV-specific miRNAs, in silico prediction was carried out to determine if the ASFV genomes of two different ASFV strains with different degrees of virulence, encode possible miRNA precursors. Subsequently, to explore whether the possible miRNAs candidates are present in the ASFV, small RNA libraries from target tissues were constructed and analyzed.The Vmir prediction algorithm  was used4 hemadsorbing units (HAU50), was used for each virus. One non-infected pig was included as negative control. All samples used are summarized in Table In order to explore whether these candidates were present in the ASFV genome, samples from spleen and submandibular lymph node (SLN) from seven 8-week-old Landrace X Large White male pigs were used. The animals were acquired from Hypor , and were fed ad libitum with conventional compound feed for growing pigs. The animals were intramuscularly inoculated in the neck with the virulent ASFV strains: BA71 or E75 or with the attenuated E75CV1, obtained by adaptation of E75 after three passages in Vero cell lineage. The same dose, 10The time points chosen for each experimental infection were selected taking into account previous results  and coveAll animal experiments were carried out in the BSL3 facilities at CReSA, and all animal managements were performed in accordance with the guidelines of the Good Experimental Practices. All procedures were approved by the Ethical and Animal Welfare Committee of the Universitat Autònoma de Barcelona with the permit number DMAH-5796.SYBR Green quantitative PCR (qPCR) was used with the following oligonucleotides: pF1 primer (5′ CCTCGGCGAGCGCTTTATCAC 3′) and pR2 (5′ GGAAACTCATTCACCAAATCCTT 3′), designed in a highly conserved region within the p72 ORF . qPCR miTotal RNA extractions were carried out in order to construct small RNA libraries from spleen and SLN samples from the animals infected with E75CV1 necropsied at 31 dpi, the E75CV1-infected animals and re-inoculated at 31 dpi with BA71, necropsied at 38 dpi, and from one non-infected animal as described in  with somSus scrofa genome were analyzed in [Strict quality parameters of the individual nucleotide sequence reads has been applied and only those with the Phread base quality scores of Q > 30 in all its bases have been used. Primer sequences were removed and only those insert sequences between 15 and 29 nucleotides were used for further analysis. Sequences aligned with lyzed in  since thlyzed in  was usedlyzed in .The in silico prediction allowed the identification of 415 miRNA candidates in the E75 strain with a score higher than 100 Fig.  and in tIn the animal infection, the pigs inoculated with the virulent strains E75 and BA71 developed typical clinical signs, while the pigs inoculated with the attenuated E75CV1 strain developed mild or no clinical signs . QuantitTotal RNAs from SLN and spleen were used to construct small RNA libraries from the selected animals. High-throughput sequencing provided 272,967 reads from the eight new libraries that, in addition to eight previously sequenced libraries with 301,802 reads, provided a total of 574,769 reads from the sixteen libraries  and other human neurotropic herpesviruses (HSV1 and HSV2) , which dIn this study, we have found that ASFV, under these experimental conditions, does not express viral miRNAs like other viruses such as Cowpox virus or Porcine circovirus type 2 (PCV2) , 35. IntIn the present work, we can conclude that African swine fever virus does not express viral miRNAs in the in vivo conditions studied here. This finding is contrary to what happens with other large DNA viruses and can be relevant to the biology of this important pathogen.


Consistent with all patient results, the prognosis of patients with higher VNN3 expression was worse in both low stage (I and II) and high stage (III and IV) . Area under the curve and receiver operating characteristic curves supported our results that highlighted VNN3 expression as a suitable ccRCC biomarker. Multivariate analysis also verified the prognostic performance of VNN3 expression . Altogether, we suggest that VNN3 is applicable as a new biomarker to establish prognosis in patients with ccRCC.Although pathological observations provide approximate prognoses, it is difficult to achieve prognosis in patients with existing prognostic factors. Therefore, it is very important to find appropriate biomarkers to achieve accurate cancer prognosis. Renal cell carcinoma (RCC) has several subtypes, the discrimination of which is crucial for proper treatment. Here, we present a novel biomarker, VNN3, which is used to prognose clear cell renal cell carcinoma (ccRCC), the most common and aggressive subtype of kidney cancer. Patient information analyzed in our study was extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. VNN3 expression was considerably higher in stages III and IV than in stages I and II. Moreover, Kaplan–Meier curves associated high VNN3 expression with poor prognoses (TCGA,  Renal cell carcinoma (RCC) is the 6th most common cancer in men worldwide, accounting for about 5% of all oncology-diagnosed cases, and the 10th most common cancer in women with a rate of about 3%  in March 2018. Patients with insufficient clinicopathological data were excluded. Overall, 58% of patients in TCGA (n = 262) and 66% of patients in ICGC (n = 60) were classified as low stage (I and II). Approximately 57% of the patients in both cohorts were male. Detailed patient characteristics are summarized in Clinical and genomic data for TCGA and ICGC cohorts were downloaded from the ICGC data portal  and high stage (III and IV) in both cohorts were identified via the Wilcoxon signed-rank test using the ‘coin’ package.We performed survival analysis to predict the overall survival (OS) of TCGA and ICGC cohorts. In the survival curves (Kaplan–Meier), we determined a cut-off value that had the maximal Uno’s C-index using a previously described five-fold cross-validation method  and ICGC (p = .017) cohorts.We evaluated the possibility of VNN3 as a prognostic marker in ccRCC by analyzing Kaplan–Meier curves that compared the relationship between VNN3 expression level and patient survival. Notably, patients with low VNN3 expression showed a higher survival rate than those with high VNN3 expression in TCGA  when compared to TCGA  and the antioxidant, cysteamine (Mariani and Roncucci Unlike the other vanins, VNN3 appears to encode a secreted protein (Mariani and Roncucci


Borrelia burgdorferi) and babesiosis (Babesia microti) is the Ixodes scapularis tick, which has been documented in 77 of 92 Indiana counties  (B. microti immunoglobulin G (IgG) or immunoglobulin M (IgM) antibodies by enzyme immunoassay (EIA) at a commercial laboratory. To further evaluate these findings, ISDH and CDC conducted a laboratory investigation using specimens from some of the patients.In the midwestern United States, the principal vector for Lyme disease  for total immunoglobulin to B. microti.The physician in northwestern Indiana was asked to select clinically representative patients for further investigation; 14 were chosen, including five of the eight who had positive  results . Whole bThe 14 patients lived in seven northwestern Indiana counties. The median age was 46 years (range = 10–76 years); nine were female . The onlB. microti IFA titers of 1:64; they reportedly became symptomatic in July 2015 and June 2016, respectively. The results of all other Babesia testing were negative.Patient specimens were collected a median of 172 days (range = 22–348 days) after reported illness onset dates; the interval was ≥3 months for all but two patients (D and G). One patient (M) had positive C6 peptide EIA results; no patient had positive whole cell sonicate EIA or immunoblot results . All patBabesia infection, and only two had serologic evidence of B. microti infection. A B. microti IFA titer of 1:64 is insufficient laboratory evidence to fulfill the national surveillance case definition for non–transfusion-associated babesiosis (This laboratory-based investigation does not suggest a cluster of Lyme disease or babesiosis cases among these patients. None had serologic evidence of Lyme disease or parasitologic or molecular evidence of I. scapularis ticks in areas where the pathogens are present. Physicians in low-prevalence states can increase the positive predictive value of laboratory testing by carefully selecting patients for testing, following established diagnostic recommendations, and using certified or accredited laboratories (Lyme disease and babesiosis should be considered in the differential diagnosis for patients with clinically compatible illness and potential exposure to


Between 28 June and 17 September 2018, 27 cases of human West Nile virus infections were recorded in Austria; four cases of West Nile neuroinvasive disease, 11 cases of West Nile fever, six infections detected by blood donation screening and six imported cases. In addition, 18 cases of human Usutu virus infections  were recorded. This is the highest number of annual infections recorded in Austria since the introduction of both viruses. Flavivirus; family Flaviviridae). WNV infection in humans may result in disease of varying severity, from West Nile fever (WNF) to possibly lethal West Nile neuroinvasive disease (WNND) [West Nile virus (WNV) and Usutu virus (USUV) are closely related mosquito-borne viruses (genus e (WNND) . Human ie (WNND) ; severe e (WNND) ,4. In 20The first three human West Nile disease (WND) cases were identified in eastern Austria in 2009 and 2010; two cases of WNND and one case of WNF . Since tBetween 28 June and 17 September 2018, 27 cases of human WNV infections were recorded of which 21 were locally acquired. Four cases were WNND, 11 cases were WNF, six were WNV infections detected by blood donation screening and six cases were associated with travels to Serbia (n = 2), Italy (n = 1), Greece (n = 1), Hungary (n = 1), and Croatia (n = 1). USUV infections were identified in 18 of 31,598 blood donations tested . All autIn Austria, human, veterinary and entomological surveillances to detect WNV and USUV include seasonal testing of blood donations originating from endemic areas, detailed sample examination of humans and equids with neurological symptoms, passive ornithological monitoring as well as regular, nationwide mosquito surveillance (for details see ).In 2014, the Blood Service for Vienna, Lower Austria and Burgenland of the Austrian Red Cross introduced an automated nucleic acid test (NAT) on the cobas 8800 system  for all blood donated between 1 June and 30 November each year. Already in the first year of screening, one WNV-positive donation was identified ,8 as welIn 2018, overall, 15 patients and three blood donors showed signs and symptoms compatible with WNV infection . Three oSequencing of short fragments of the WNV C/prM (294nts) and USUV NS5 396nts) genes enabled exact identification of the detected viruses. This analysis revealed that all WNV strains belonged to lineage 2 with a 98.98–100% identity to the Austrian strain BD2/2016 [nts genesWith 21 locally-acquired and six imported human WNV infections the 2018 transmission season has been the most relevant since the introduction of the aetiological lineage 2 WNV to Austria in 2008 ,11. The One blood donor was found to have a double infection with both WNV and USUV for the first time. While the central European lineage 2  was the USUV is spreading in Europe, which may lead to increased numbers of human infections. In countries with blood donation testing for flavivirus RNA, health authorities should be aware that positive WNV screening results could be due to USUV infections and have to be further differentiated. In addition, countries where blood donation testing is not performed for flaviviruses, USUV might be transmitted through contaminated blood units. Although no transfusion-associated USUV infection has been reported so far, it is of utmost importance to further investigate the clinical relevance of USUV infections.


Urinary copy numbers of the mitochondrial DNA (mtDNA) genes cytochrome-c oxidase-3 (COX3) and nicotinamide adenine dinucleotide dehydrogenase subunit-1 (ND1) were measured. Urinary mtDNA levels were elevated in the IgAN group compared with that in HVs (p < 0.001). Urinary ND1 levels were significantly higher in the low proteinuria group than in the high proteinuria group (p = 0.027). Changes in urinary levels of ND1 and COX3 were positively correlated with changes in proteinuria  and inversely correlated with changes in the estimated glomerular filtration rate  after medical treatment. Mitochondrial injury played important roles in IgAN pathogenesis and may be involved in early-stage glomerular inflammation, prior to pathological changes and increased proteinuria. The correlation between changes in urinary mtDNA and proteinuria suggest that these factors may be promising biomarkers for treatment outcomes in IgAN.Mitochondrial injury plays important roles in the pathogenesis of various kidney diseases. However, mitochondrial injury in IgA nephropathy (IgAN) remains largely unexplored. Here, we examined the associations among mitochondrial injury, IgAN, and treatment outcomes. We prospectively enrolled patients with IgAN and age-/sex-matched healthy volunteers (HVs) as controls ( Within 20 years after diagnosis, one-fourth of patients will progress to end-stage renal disease (ESRD), and an additional 20% will have a gradual decline in renal function.IgA nephropathy (IgAN) is a type of mesangial proliferative glomerulonephritis (GN) characterized by diffuse deposition of IgA in the glomerular mesangium1. Renal biopsy is indispensable for diagnosis and prediction of prognosis4. However, because renal biopsy is an invasive diagnostic method and cannot be performed periodically, there are limitations to validating treatment outcomes and predicting the prognoses. Moreover, there is a growing interest in identification of biomarkers as a method to complement the limitations of renal biopsy5.Although the data are not always consistent, hypertension (HTN), proteinuria, reduced glomerular filtration rate (GFR), and unfavorable histopathological findings at the time of diagnosis have all been shown to be associated with poor prognosis in IgAN7, acute kidney injury (AKI)8, chronic kidney disease (CKD)10, and obesity-related hyperfiltration11. Data are inadequate regarding the relationship between IgAN and mitochondrial injury. Cases of patient with mitochondrial disease and presence of a mutation in the mitochondrial gene accompanied by IgAN were reported13. Mitochondrial genetic variants in patients with IgAN and ESRD were investigated in a previous study14. Acquired factors associated with kidney diseases, such as oxidative stress15, and ischemia/hypoxia16, renin-angiotensin-aldosterone system activation18, proteinuria21, cause mitochondrial dysfunction. Mitochondrial dysfunction induces podocyte injury, tubular cell damage, and endothelial cell damage22. When mitochondrial injury occurs, fragments of mitochondrial DNA (mtDNA) are released into the cytosol from the matrix and then enter into the systemic circulation24. In addition, mitochondrial injury in the kidney may result in the release of these fragments into the urine. Previous studies have shown that urinary mtDNA copy numbers can be considered surrogate markers of mitochondrial injury in various kidney diseases27.The kidney has high energy demand and is rich in mitochondria. Moreover, mitochondrial dysfunction plays an important role in the pathogenesis of various kidney diseases, including focal segmental glomerulosclerosisAccordingly, we hypothesized that the pathogenesis of IgAN may be associated with mitochondrial injury and that urinary mtDNA may serve as a valuable biomarker for evaluating treatment outcomes and predicting prognosis in patients with IgAN. In this study, we examined whether urinary mtDNA was elevated in patients with IgAN and assessed correlation with treatment outcomes and existing prognostic markers.Table 10 nicotinamide adenine dinucleotide dehydrogenase subunit-1 (ND1)/nDNA and log10 cytochrome c oxidase subunit 3 (COX3)/nDNA were elevated in the IgAN group compared with those in HVs  and COX3  levels did not correlate with urinary KIM-1 levels. Changes in ND1  and COX3  urinary levels also did not correlate with changes in KIM-1 levels 6 months after medical treatment. Urinary KIM-1 levels were positively correlated with baseline proteinuria levels  and were related to endocapillary proliferation according to the Oxford classification . KIM-1 levels were significantly reduced after medical treatement  level were significantly higher in the IgAN group than in HVs . All 19 patients were treated with angiotensin receptor blockers (ARBs), and six were treated with an immunosuppressive agent . Medical treatment did not reduce urinary levels of ND1 and COX3 . Baseline proteinuria levels were lower (p = 0.029) and urinary ND1 and COX3 levels were higher  in patients treated only with ARBs (only ARB treatment group) than those treated with ARBs and immunosuppressant (concurrent treatment group) in IgAN patients. Urinary levels of ND1 and COX3 were not reduced 6 month after treatment in concurrent treatment group  and only ARBs treatment group .Urinary mtDNA in 19 patients in the IgAN group was measured 6 months after medical treatment. In followed patients, urinary ND1 and COX3 showed positive correlations with changes in proteinuria  at 6 months and were inversely correlated with changes in eGFR at 12 months  and COX3 (p = 0.661) levels between the response and no response groups . Interestingly, when dividing the IgAN group into two subgroups according to baseline proteinuria, a subgroup of patients with lower proteinuria showed higher urinary mtDNA levels. In the same context, because of more patients with high proteinuria were treated with immunosuppressant, only ARB treatment group showed lower baseline proteinuria levels compared with concurrent treatment group. However, baseline urinary mtDNA levels were higher in only ARB treatment group than concurrent treatment group.We also analyzed correlations between urinary mtDNA and existing clinical and histopathological prognostic markers at presentation in IgAN and found that there were no correlations. These findings are not consistent with previous studies in AKI, HTN, and diabetes mellitus (DM) nephropathy31. In our patients with IgAN, we found that urinary mtDNA copy numbers were higher in patients with less histological damage and proteinuria. These results may indicate that mitochondrial damage is involved in the early stage of glomerular inflammation and occurs prior to pathological changes. Accordingly, increased proteinuria and reduced renal function and urinary mtDNA may be the earliest signs of kidney injury. However, the duration of our study was relatively short; thus, further studies are needed to confirm these findings as potential prognostic markers in patients with IgAN.According to our current knowledge, the immunopathogenesis of IgAN involves a multi-hit pathogenic process. The presence of increased amounts of underglycosylated IgA1 induces autoantibodies and the formation of immune complexes, which activate the complement pathway and contribute to mesangial activation and proliferation. Glomerular inflammation results in podocyte injury, proteinuria, tubulointerstitial inflammation, and fibrosis11. In contrast, KIM-1 levels decreased after medical treatment in our IgAN cohort, consistent with previous findings32. KIM-1 is a marker of tubule injury in the kidney; however, urinary mtDNA could have been derived from all cells in the nephron and glomerulus. These different sources could explain the lack of changes in mtDNA copy numbers in IgAN. Alternatively, urinary mtDNA copy numbers may reflect overall kidney cell damage and could be used as a unique renal injury marker distinct from existing renal injury markers.Medical treatment for IgAN did not affect mtDNA copy numbers. Notably, body weight reduction after bariatric surgery did not decrease urinary mtDNA copy numbers in a previous study31. Moreover, a recent meta-analysis also showed that reduced proteinuria is associated with kidney outcomes, including doubling of SCr, ESRD, or death, and could be used as a surrogate endpoint in trials of IgAN33. Accordingly, it seems reasonable to conclude that reduced proteinuria is associated with improved kidney outcomes. In our study, percent reduction in urinary mtDNA copy numbers was found to be related to improved eGFR and decreased proteinuria. These findings suggest that urinary mtDNA copy numbers could also be used as a surrogate marker of kidney outcomes in IgAN. Taken together, these results suggest that alleviation of mitochondrial injury through preventive and therapeutic measures may improve clinical outcomes in IgAN, particularly at an early stage.Changes in urinary mtDNA levels were correlated with changes in eGFR and proteinuria levels in our study. Substantial observational data have supported that reduced proteinuria is associated with improved renal survivalCOX3 and ND1 encode the enzyme responsible for the mitochondrial respiratory chain and the electron transport, respectively. Previous studies showed that ND1 did not differ from COX3 copy numbers in obese African American hypertensive patients34 and upon chronic renal injury in hypertensive patients25. However, urinary COX3 copy numbers from obese patients with glomerular hyperfiltration were not elevated compared with HVs11. In this study, urinary ND-1, but not COX3 levels were higher in the low proteinuria group compared with the high proteinuria group. There are various factors that cause mitochondrial injury in kidney disease. We think the causes of mitochondrial injury may be different for each kidney disease, leading to differences in mtDNA injury patterns. Although there was no statistical significance, urinary COX3 levels tended to be higher in the low- vs. high-proteinuria, and given the small sample size, ND-1 is unlikely to be a better mitochondrial injury marker than COX3.To our knowledge, there has been no study evaluating the urinary mtDNA levels in GN. Since we have evaluated only patients with IgAN, it is not clear whether elevated urinary mtDNA levels is a ubiquitous finding in GN or a unique finding in IgAN. To clarify this, future studies are needed to measure and compare the urinary mtDNA in each GN.36, CKD25, and obesity-related hyperfiltration11. Age-/sex-matched HVs were collected to minimize the effects of other factors that could increase circulating mtDNA levels. However, patients with IgAN tended to be older and have higher body mass index than HVs. An important limitation of this study is that we could not measure circulating mtDNA levels, due to which we cannot exclude the possibility that urinary mtDNA levels were elevated due to increased circulating mtDNA levels by other factors in the urine. In addition, although urinary mtDNA was increased in patients with IgAN compared with HVs, the variation in IgAN group was so high that it was difficult to detect individual differences among patients. This limitation could be compensated by measuring circulating mtDNA. Finally, we could not explain the specific mechanisms through which mitochondrial injury leads to kidney damage or determine in which part of the kidney mitochondrial injury may occur. In early stage of IgAN pathogenesis, galactose-deficient IgA1-containing immune complexes activate mesangial cells that secrete components of extracellular matrix, releasing various mediators including proinflammatory cytokines39 and activating renin-angiotensin-alodosterone system42. These events may eventually lead to oxidative stress43 and mitochondrial dysfunction. Further studies are required to support this hypothesis. In this study, we identified ultrastructural changes in the mitochondria through kidney biopsy specimens of two patients with IgAN. However, we could not quantitatively measure the extent of damage and determine in which part of the kidney mitochondrial injury may occur. In future studies, if ultrastructural changes in the mitochondria of kidney biopsy specimen from patients with IgAN is measured quantitatively using electron microscope and compared with urinary mtDNA level, more accurate mitochondrial injury site could be determined.There were some limitations to our study. First, the sample size was small, and the duration of follow-up was short, which may have made the study underpowered for evaluating whether differences in urinary mtDNA levels were useful for predicting treatment responses or prognosis. Attenuated urinary mtDNA levels were correlated with decreased proteinuria and improved eGFR in our study; however, these findings should be confirmed further in additional studies with larger sample sizes and longer follow-up durations. Second, we did not measure serum mtDNA copy numbers. We measured urinary mtDNA levels, because previous studies showed that, unlike circulating mtDNA levels, urinary mtDNA levels are correlated with renal dysfunction and clinical outcomes in various kidney disease including AKIIn conclusion, our findings suggested that mitochondrial injury may play a role in the pathogenesis of IgAN and may be involved in early-stage glomerular inflammation prior to pathological changes and increased proteinuria. Mitochondrial injury may be associated with changes in proteinuria and eGFR and may therefore represent a promising biomarker for treatment outcomes in patients with IgAN.n = 31) who were collected from the Cohort for Biomarker Inquiry of Renal Aggravation (COBRA) cohort at Soonchunhyang University Seoul, Bucheon, and Cheonan Hospitals from May 2016 to January 2018. Age- and sex-matched HVs were enrolled as controls (n = 31). HVs were included if they had no history of DM, HTN, congestive heart failure, coronary artery disease, and stroke and did not take any medications. All participants provided written informed consent.This was a multicenter prospective cohort study. The current study was conducted according to the principles outlined in the Declaration of Helsinki, and clinical data from patients were obtained after approval of the study by the Institutional Review Board (IRB No. 2016-01-002-007) of Soonchunhyang University Hospital. We enrolled patients with biopsy-proven pure IgAN . We also measured urinary ND1 and COX3 levels 6 months after medical treatment in IgAN group (n = 19). DNA was isolated from urine samples (1.75 mL) using DNA isolation kits from Norgen Biotek . We analyzed DNA concentrations using a NanoDrop Spectrophotometer . RT-quantitative PCR (qPCR) was performed using the ND1 primers (forward 5′-AGTCACCCTAGCCATCATTCTACT-3′ and reverse 5′-GGAGTAATCAGAGGTGTTCTTGTGT-3′) and COX3 primers (forward 5′-AGGCATCACCCCGCTAAATC-3′ and reverse 5′-GGTGAGCTCAGGTGATTGATACTC-3′), obtained from Thermo Fisher Scientific, and 20 ng template DNA/sample. The PCR conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 60 s.Urinary copy numbers of the mtDNA genes 11.mtDNA copy numbers were corrected to the nuclear control gene RNAse-P  using human genomic DNA for the standard curve. mtDNA copy numbers were calculated using Copy Caller software (Thermo Fisher Scientific) and expressed as a mtDNA/nDNA ratio46. We measured KIM-1 levels in the IgAN group and HVs at the time of kidney biopsy and in the IgAN group at 6 months after medical treatment. A single investigator performed all measurements blinded to the clinical information.KIM-1 levels were measured using enzyme-linked immunosorbent assays with a commercial kit  according to the manufacturer’s protocol and standardized with urine creatinine levels (ng/mg), as previously described3 pieces using a scalpel. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a digital electron microscope . All glomeruli were examined at x3000 and examined at a higher magnification for mitochondria structure47.We examed structural changes in the mitochondria in two patients with IgAN using transmission electron microscopy. A kidney biopsy specimen from a living kidney donor who had normal kidney function was assessed as a control. To detect mitochondrial ultrastructure morphology, the kidney samples were cut into 1 mm2 tests or Fisher’s exact tests for categorical variables, as appropriate. Urinary mtDNA copy numbers were log-transformed. Spearman’s rank correlation coefficient was used to analyze the relationships between urinary mtDNA copy numbers and clinical and histological variables. All statistical tests were two-sided, and the results were presented with 95% confidence intervals. We considered p values of less than 0.05 to indicate statistical significance. All analyses were performed using SPSS 25 for Windows .Descriptive characteristics of the study population were reported as means ± standard deviations or medians with interquartile ranges for continuous variables and as frequency counts with percentages for categorical and binary variables. Comparisons of differences between groups were made using Mann-Whitney and Wilcoxon signed rank tests for continuous variables and either χ