This was most striking in patients with nuclear TYMP or mitochondrial m.3243A>G MT-TL1 mutations, in keeping with differences in mitochondrial density seen postmortem. Some regional binding patterns corresponded to clinical presentation and underlying mutation, even in the absence of structural changes on MRI. This was most obvious for the cerebellum, where patients with ataxia had decreased binding in the cerebellar cortex, but not necessarily volume loss. Overall, there was a positive correlation between aberrant [11C]PK11195 binding and clinical severity.Forty-six percent of symptomatic patients had volumes of abnormal radiotracer binding greater than the 95th percentile in controls. [These findings endorse the use of PET imaging with TSPO radioligands as a noninvasive in vivo biomarker of mitochondrial pathology.This study provides Class III evidence that brain PET-MRI with TSPO radioligands identifies mitochondrial pathology. This study provides Class III evidence that brain PET-MRI with TSPO radioligands identifies mitochondrial pathology.Patients with genetically confirmed mitochondrial disease were recruited through a specialist mitochondrial disease clinic at Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, United Kingdom. Fifteen patients consented to undergo PET-MRI. The scan of patient 13 was cancelled because of an upper respiratory tract infection and this patient was excluded from the analysis. Human phenotype ontology terms for each patient were assigned based on electronic health records. Modified Rankin Scale (mRS) score and Scale for the Assessment and Rating of Ataxia (SARA) were calculated based on available clinical examinations in the electronic health records. The research protocol was approved by a National Research Ethics Service committee (REC ID: 16/LO/2093). The healthy control cohort comprised subjects from projects in depression  and Parkinson disease  that used the same data acquisition protocol on the same PET-MRI scanner. All these projects received approval from the Administration of Radioactive Substances Advisory Committee and all participants provided written informed consent in accordance with the Declaration of Helsinki.11C]PK11195 was injected over approximately 30 seconds and list-mode PET data were acquired for 75 minutes. The median (interquartile range [IQR]) injected activity was 397 (62) MBq with corresponding injected mass values of 3.8 (3.8) μg. Injected activity per unit body weight (MBq/kg) and injected mass were not significantly different between the mitochondrial disease and control groups. Attenuation correction included the use of a multisubject atlas method21 and improvements to the MRI brain coil component.22 Other data corrections  were as implemented on the scanner. Dynamic sinograms were reconstructed into 128 × 128 × 89 arrays (2.0 × 2.0 × 2.8 mm voxel size) using time-of-flight ordered subsets expectation maximization,23 with 6 iterations, 16 subsets, and no smoothing. Brain T1 and T2 MRI acquired as part of the protocol were reviewed as part of routine clinical radiology reporting.All participants underwent PET and MRI in a single session on a GE Signa PET-MR scanner , which can simultaneously acquire PET and 3T MRI data. PK11195 binding in large parts of the brain in individual patients.Across patients and different regions of the brain, the volume of significantly increased or reduced BP11C]PK11195 binding in specific brain regions would be preferentially affected by mitochondrial dysfunction, BPND was determined in 12 white matter , while BPND in the hippocampus (p = 0.002) and substantia nigra (p = 0.001) were significantly increased  (ND (p = 0.001) compared to controls  . Subgroucontrols . This wacontrols , suggestND in patients with mitochondrial disease was mostly driven by 3 individuals  whose white matter BPND was severely reduced (see MT-TL1 (m.3243A>G) mutation; patient 11 had mitochondrial neurogastrointestinal encephalopathy (MNGIE) caused by compound heterozygous TYMP mutations  between mRS and the fraction of voxels across all ROIs (gray and white matter) with a significant change (increase or decrease) in [11C]PK11195 binding (MT-TL1 mutation or the m.14487T>C MT-ND6 mutation), where clinical symptoms as measured by mRS were always worse in those patients with a higher fraction of voxels with changed binding (doi.org/10.5061/dryad.zs7h44j7s). For those patients with ataxia syndromes , this trend was most striking for signal changes in the cerebellum, where a higher voxel fraction with aberrant [11C]PK11195 binding was associated with worse performance on the SARA scale (doi.org/10.5061/dryad.zs7h44j7s). This demonstrates that, despite high interindividual variability in both radiotracer binding and clinical presentation, CNS binding of [11C]PK11195 is related to clinical severity.We used the mRS to measure clinical disability in the 13 patients with symptomatic mitochondrial disease and the SARA binding : patient11C]PK11195 PET brain imaging of the mitochondrial outer membrane protein TSPO in a group of representative patients with genetically confirmed mitochondrial disease. Our results demonstrate a correlation between the degree of abnormal radiotracer binding across the brain and clinical severity in symptomatic patients. In addition, we found that regional binding abnormalities may correspond to clinical presentation, are mutation-specific, and mostly occur in the absence of obvious structural changes on brain MRI.We describe [11C]PK11195 PET imaging of TSPO is widely used as a marker of neuroinflammation, due to its accumulation in activated microglia in the brain.16-18 Although this is probably valid for CNS disorders where the primary disease process is related to inflammation, TSPO abundance and radiotracer binding are likely to be differentially affected in many other noninflammatory conditions.19 In particular, a decrease in radiotracer binding, as previously observed in autism spectrum disorder,20 schizophrenia,31 or inactive multiple sclerosis lesions,32 is more difficult to reconcile with microglial activity or inflammation. Given its localization to mitochondria in all cell types of the CNS,15 it is conceivable that TSPO abundance in the brain is affected by pathologic changes associated with mitochondrial disease.[11C]PK11195 radiotracer binding across the brain exceeded the 95th percentile of the control population. In contrast to neuroinflammatory disorders, radiotracer binding was not simply increased, but patients and brain regions had variable patterns of reduced or enhanced BPND. This indicates that mitochondrial disease is not necessarily accompanied by inflammation and microglial activity. These findings are in line with previous neuropathologic studies that only showed microglial proliferation in active stroke-like lesions in the temporal and occipital cortex of patients with MELAS,33 or late stages of Leigh syndrome lesions,34 but not elsewhere in the brain.36The 14 patients with mitochondrial disease had mutations in the mitochondrial or nuclear genomes causing a range of mitochondrial syndromes at various stages of their disease course , thus pr37 and model organisms39 where genetic perturbation of mitochondrial function was associated with a reduction in mitochondrial density in the distal axons of neurons. In gray matter, where neuronal cell bodies are located, binding was more variable and frequently increased. Interestingly, in the soma of dopaminergic neurons of the substantia nigra, a gray matter region in the midbrain where we observed a significant increase in average BPND  imaging.29 Interestingly, many of our patients had large regions of asymmetric binding abnormalities, with otherwise normal or symmetric MRI. In the cerebellum of patients with cerebellar ataxia, we found significant reductions in [11C]PK11195 binding using regional BPND values corrected for atrophy with GTM partial volume correction. Although many patients had some degree of cerebellar atrophy, reduced binding was even observed in the absence of overt cerebellar atrophy as reported on MRI. Postmortem studies in patients with ataxia due to mitochondrial disease have found a reduction in mitochondrial density in the cerebellar cortex, possibly related to microscopic neuronal loss.48 Together, these findings suggest that changes in [11C]PK11195 binding may precede structural MRI abnormalities and could be more sensitive than MRI at detecting reduced neuronal or mitochondrial density.CNS involvement in metabolic disorders such as mitochondrial disease is often thought to be bilateral and symmetric, with generalized brain or cerebellar atrophy, widespread white matter changes, or bilateral, symmetric basal ganglia lesions.11C]PK11195. Compared to higher-affinity second-generation TSPO PET tracers, such as [11C]PBR28 and [11C]DPA-713, [11C]PK11195 has lower transport into brain tissue and a lower ratio specific to nonspecific binding (except for low affinity binders), resulting in BPND estimates of inferior statistical quality.49 However, [11C]PK11195 has the advantage of obviating the need to divide the participants into binding affinity groups  driven by a polymorphism in the TSPO gene.50 In a small cohort, such as in this study, avoiding this subdivision is particularly advantageous. However, if much larger, multicenter studies employing TSPO PET imaging are conducted in the future on patients with mitochondrial disease, the cost, access to tracers or equipment, and reproducibility of imaging in different settings all will have to be taken into account, and the increased sensitivity offered by more accessible and cost-effective second-generation TSPO PET tracers labeled with fluorine-18 may be considered.This study used the prototypic TSPO PET tracer [11C]PK11195 BPND was quantified using reference tissue kinetic modeling, which requires a key additional assumption compared to gold standard kinetic modeling with an arterial input function; namely, that the reference tissue input is devoid of specific binding. Reference tissue inputs produced by the method employed in this study  were shown to have lower total distribution volumes than those obtained from the most commonly used anatomical reference tissue, the cerebellum.27 This supports the notion that reference tissue inputs from supervised cluster analysis better approximate the ideal situation of a null contribution from specific binding. To further improve the accuracy of the BPND estimates, the reference tissue model incorporates correction for vascular signal, including binding to endothelial TSPO receptors.26 Although reference tissue modeling of [11C]PK11195 PET data with a reference tissue input from supervised cluster analysis has been validated with arterial input function kinetic modeling,51 ideally this validation process should be extended to patients with mitochondrial disease.PK11195 binding abnormalities than their less disabled counterparts. Our cohort was too small and heterogeneous to demonstrate strong correlations between specific symptoms and regional binding characteristics. However, for patients with ataxia, symptom severity was clearly related to reduced radiotracer binding in the cerebellum. Larger natural history studies are needed that measure disease progression together with whole-brain and region-specific radiotracer binding, including in genetically homogeneous patient cohorts, to confirm the validity of TSPO PET as a reliable biomarker for interventional studies in thus far incurable mitochondrial diseases.Research into novel therapies of mitochondrial disease has been hampered by the lack of useful noninvasive biomarkers that measure disease severity and progression.


Tumor necrosis factor alpha (TNF-α) plays important roles in processes such as immunomodulation, fever, inflammatory response, inhibition of tumor formation, and inhibition of viral replication. TNF-α and its receptors are ubiquitously expressed in developing organs and they regulate the survival, proliferation, and apoptosis of embryonic stem cells (ESCs) and progenitor cells. TNF-α is an important inflammatory factor that also regulates the inflammatory response during organogenesis, and its cytotoxic effects can interfere with normal developmental processes, even leading to the onset of diseases. This review summarizes the various roles of TNF-α in organogenesis in terms of its secreting pattern, concentration-dependent activities, and interactions with other signaling pathways. We also explored new potential functions of TNF-α. Tumor necrosis factor alpha (TNF-α) belongs to the TNF superfamily  of proteThis review summarizes the roles of TNF-α in the development of various organs and associated diseases. We also highlight the mechanisms of TNF-α from multiple aspects, including its effects on stem cells/progenitor cells, secreted forms, concentration-dependent activities, and interactions with other signaling pathways.The effects of TNF-α can be traced back to gastrulation, during which it promotes embryonic differentiation and cell apoptosis . Organogin vivo and in vitro , progenitor cells, and differentiated cells. It not only inhibits the self-renewal of mouse ESCs but also induces their apoptosis and inhibits their differentiation into embryos . TNF-α pin vitro .Tumor necrosis factor alpha is also involved in regulating the proliferation, apoptosis, and differentiation of progenitor cells. It promotes the proliferation and differentiation of neuroblasts into astrocytes in the human fetal cortex , inhibitAlthough transmembrane TNF-α (pro-TNF) is biologically active, TNF-α primarily exerts autocrine and paracrine functions in a soluble, trimeric form during developmental processes. TNF-α acts as an autocrine and paracrine growth factor that stimulates the proliferation of hematopoietic cells and B cells . AutocriLow concentrations of TNF-α tend to promote cell proliferation, whereas high concentrations tend to inhibit cell proliferation and even induce the apoptosis of neural stem cells/progenitor cells in the subventricular zone of neonatal mice, and of osteoclast progenitor cells and intestinal stem cells in fetal mice . The bioTumor necrosis factor alpha interacts extensively with fibroblast growth factor (FGF) family, Wnt family, and TGF-β superfamily members to co-regulate developmental processes. It promotes angiogenesis by inducing basic FGF and FGF-1 expression in endothelial cells .Tumor necrosis factor alpha inhibition promotes the functional recovery of nerves by activating the Wnt3a signaling pathway in BMSCs . TNF-α iTumor necrosis factor alpha regulates the differentiation of osteoblasts by affecting the bone morphogenetic proteins (BMP) signaling pathway . It alsoTumor necrosis factor alpha induces the expression of hepatocyte growth factor in human bone marrow- or adipose-derived progenitor cells and in MSCs to promote tissue growth and repair . TNF-α hin vivo and in vitro and negatively affects brain development in vivo  or surrounding cells . TNF-α tends to promote cell proliferation at low concentrations and inhibit cell proliferation and induce apoptosis at high concentrations. TNF-α interacts extensively with the FGF, Wnt, and TGF-β signaling pathways to co-regulate organogenesis. It is also an important inflammatory factor that regulates the inflammatory response and exerts cytotoxic effects. The overwhelming cytotoxic effect of TNF-α during organogenesis due to inflammation interferes with normal developmental processes and can trigger disease onset. Despite considerable knowledge about TNF-α and its functions in organogenesis, several questions remain. For example, how TNF-α prevents the activation of undesirable immune responses during developmental processes remains obscure. Low concentrations of TNF-α promote the development of intestinal epithelium without triggering inflammatory response , but wheKY was in charge of acquisition of reference articles and critical revision of the manuscript for important intellectual content. HG and ZY were in charge of critical revision of the manuscript for important intellectual content. XX was in charge of drafting of the manuscript and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates . Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen. Serologic assays are routinely used for evidence of exposure to pathogens, typically by assessing antibodies to a specific antigenic epitope to the pathogen of interest in blood samples collected from individuals or populations of humans or animals . An ideaThe phenomenon of nonspecific binding in solid-phase immunoassays has been recognized and is associated with primary or secondary antibody binding to nontarget antigens or surfaces, high immunoglobulin levels, or inflammatory markers ,2. IdentFeline immunodeficiency virus (FIV) is a T-cell tropic lentivirus with worldwide distribution in cats . FIV infWe developed standard antigen-enzyme-linked immunosorbent assay (ELISA) serologic detection assays for the assessment of exposure to FIV and FFV. Use of serum from an unvaccinated, specific pathogen-free (SPF) cat colony as negative controls and cats experimentally infected with FIV, FFV, or vaccinated with a commercially available FIV vaccine as positive controls repeatedly resulted in 100% sensitivity and specificity of our assay systems following seroconversion four weeks after viral exposure. We took advantage of a unique bioarchive containing well-characterized field and experimental serum samples to compare ELISA binding characteristics of known positive and negative individuals on plates validated with ‘gold standard’ serum samples. Our findings clearly document significantly higher nonspecific binding in samples obtained from animals with at least one other documented viral infection, particularly for FIV antigens.In total, 56 individual serum samples were tested (31 samples for FIV and 61 samples for FFV), . These ip-value 0.87, p-value < 0.001, The FIV ELISA indeterminant range optical density (OD) value was set within the range of 0.2–0.41 based upon ELISA value measurements from sera from three SPF cats that were FPV negative (Sham cats). All SPF cats had absorbance values below the indeterminant range . SPF FPVp-value 0.66, p = 0.098). The seroreactivity of field negative samples was lower on the FFV ELISA than that noted with FIV ELISA. Overall, 2 FFV negative shelter cats, 3 SPF FIV inoculated cats, and 1 private colony cat that was FFV negative had absorbance values within the indeterminate range , FIV prebleed (n = 3), and FFV prebleed (n = 2)). All SPF cats had absorbance values below the indeterminant range . Field ste range .SPF cats that were experimentally inoculated with either FIV or FFV demonstrated an increased antibody response over time, as anticipated . Cats inE. coli antibody in the blocking steps of the ELISA did not result in decreases in nonspecific binding as compared to using 2% bovine serum albumin (BSA).Attempts at enhancing specificity for field samples without reducing sensitivity by using 5% nonfat milk, 1% casein, and 1:200 anti-An FIV diagnosis is dependent upon accurate diagnostic testing. As FIV causes antibody titers to be persistently high and FIV polymerase chain reaction (PCR) is unreliable due to variation in the FIV genetic sequence, serology is the preferred method for diagnosis . AntibodFeline foamy virus (FFV) is a retrovirus belonging to the family Spumaretrovirus . The virThis study aimed to evaluate the sensitivity and specificity of in-house laboratory diagnostic ELISAs for FIV and FFV comparing sera from SPF cats with a variety of known viral infections and field samples that had been well characterized. Sera from experimentally infected cats and SPF cats or sham inoculated controls were used to set positive cutoff points and indicated 100% sensitivity and specificity after the 1-month post-inoculation seroconversion period. Cats in the process of seroconverting  had ELISA OD absorbance values that fell within or below the ‘indeterminant range’ defined for each assay.Following inadvertent infection of SPF cats with feline parvovirus or when testing field isolates from domestic cats from a variety of housing settings, we observed ELISA OD values in the indeterminant range in 22.4% of cases (13/58). We also tested SPF cats infected with FIV or FFV on the converse ELISA protocol to assess the potential for one retroviral infection to induce antibodies that cross-react with antigens from a different retrovirus. Use of SPF cat sera as a negative control and sera from cats experimentally inoculated with laboratory strains of FIV or FFV  provided an idealized parameterization of the diagnostic assays. We found that ELISA absorbance values generated from serum from cats with irrelevant viral exposures often exceeded cutoff values generated with ‘gold standard’ samples. This resulted in high numbers of indeterminant or false positive findings when analyzing field samples.The most obvious explanation for the variation seen in the background between field and laboratory cats is the SPF cats lack of exposure to field pathogens and a single exposure to a large amount of virus, resulting in a very specific antibody response with low background. Cat serum samples collected from multi-cat households or animals surrendered to shelters have the potential to be infected with multiple agents and have a much broader repertoire of polyclonal antibodies against a variety of agents, resulting in a significantly higher background. SPF cats with FFV or FPV tended to have lower cross-reactivity to FIV than samples from the private breeding colony and multi-cat household. Thus, cats with multiple pathogen exposures and ongoing infections were more likely to nonspecifically bind FIV Gag antigens than SPF cats with one viral infection. In contrast, 3 out of 12 SPF cats with FIV infection had higher seroreactivity to FFV Gag antigen and only 7.32% of FFV field samples had nonspecific binding. The higher propensity for reactivity to FIV Gag antigen than FFV Gag may be a reflection of antibody affinity or antigen preparation quality or may be a true reflection of the comparative likelihood for development of cross-reactive antibodies to these agents. The bioarchive used in this study was limited in the total number of samples as well as number of samples between groups. Testing more samples from cats with known experimental or field exposures against a battery of antigens would help to reveal more about specific mechanisms underlying cross reactive antibody production.Analysis of early timepoints in experimentally exposed animals demonstrated that early infections are a second explanation for indeterminant test results, reinforcing that paired serum samples collected 2–4 weeks apart should be able to distinguish indeterminant, nonspecific reactivity from early seroconversion. FIV vaccination produces an antibody signal over time similar to that of cats experimentally infected with FIV, leading to a false-positive FIV infection diagnosis if vaccine history is not considered . We demoE. coli antibody using the same ELISA protocol for each reagent. While only a few samples were tested, we found no difference between the blocking reagents in reducing nonspecific binding. Given that the samples tested were from cats that had either a current or previous viral infection (other than FIV), we suspect these cats have antibodies that can interact with the FIV Gag antigen.In attempts to reduce nonspecific binding on the FIV ELISA, we tested different blocking reagents including 2% BSA, 5% nonfat milk, 1% casein, and 1:200 anti-While the outcomes of this study are not surprising, use of serum from SPF and experimentally animals in comparison to field isolates provided a unique opportunity to conclusively illustrate basic assumptions about sera cross-reactivity that are not readily tested in humans or other species where control of disease exposure is feasible. Our results demonstrate that validation of FIV and other serologic assays using serum from SPF animals using standard cutoff value calculations may lead to false positives. Conversely, the use of field negative serum could result in setting thresholds that overestimate background reactivity, leading to loss of assay sensitivity. Here we used the method of three standard deviations that is a common approach to cut-point setting . In caseThis report illustrates that diagnostic assay validation using samples from individuals without exposure to antigens or pathogens may result in overestimation of sensitivity and specificity, particularly during FIV infection. Further, FIV vaccination results may result in false positive or suspect results. Setting an indeterminate range and subjecting suspect samples to additional screening tests is a potential strategy for optimizing sensitivity and specificity. Retesting paired samples 3 weeks following testing offers an option for accurate diagnosis.Giardia, Cryptosporidium, feline coronavirus (FeCOV), felis catus gammaherpesvirus 1 (FcaGHV1), and feline foamy virus (FFV). Serologic testing for FPV was conducted in 21% of the colony in 2014 and 2018 by a commercial laboratory  and using in-house qPCR (data not shown). Blood was collected via the jugular or cephalic vein without sedation. All sera collected in 2014 were negative, whereas all samples were strongly positive in 2018. SPF FPV positive cats unassigned to experimental studies (n = 6), FFV prebleeds (n = 2), FIV prebleeds (n = 3), and SPF cats exposed to sterile tissue culture fluid (MYA-1 cells or CrFK culture media) during experimental studies  were used as negative controls for both FIV and FFV ELISA tests [Negative serum samples were collected from 6 unvaccinated, specific pathogen-free (SPF) cats maintained in a breeding colony with restricted housing environment at Colorado State University. Cats were kept in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited animal facility and were routinely assessed for exposure to feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline parvovirus (FPV), calicivirus, feline herpesvirus (Rhinotr), SA tests ,10.Serum samples were collected from 3 cats infected with FIV and FFV in experimental studies at days 7, 14, 23, and 28 post-infection and days 3, 7, 10, 15, 21, 28, 56, and 70 post-infection, respectively, as previously described ,10. NegaField serum samples were obtained from 2 sources. FIV shelter positive included cats  from 2 shelter settings characterized as FIV positive by FIV virus isolation and serology . The priTwo CSU SPF cats were vaccinated with a commercially available FIV vaccine, Fel-O-Vax . Serum samples were collected at 14, 35 and 56 days post vaccination.The FIV Gag capsid antigen was prepared as previously described . The FFV2SO4 and read at 450 nm using a Multiskan® Spectrum spectrophotometer (Thermo Fisher).High binding plates  were coated with either FIV Gag or FFV Gag (100 ng/well) and incubated overnight at 4 °C. Plates were decanted, and 290 µL of 2% BSA in wash buffer were added to each well for 2.5 h at room temperature. Wash buffer  was used to remove unbound proteins from the wells. Feline serum was diluted 1:100 in ELISA diluent and added to the wells and incubated for 2 h at room temperature. Samples were run in triplicate with the average between wells determining the OD value. Wells were washed 5 times with wash buffer containing 0.2% Tween. A total of 100 µL of diluted goat anti-cat IgG peroxidase conjugate (diluted 1:5000 in ELISA diluent with 5% mouse sera) was added to each well and incubated for 1 h at room temperature and washed 5 times with wash buffer containing 0.2% Tween. Then, 100 µL of TMB were added to the wells. The reaction was stopped at 10 min with 50 µL of 2.5 N HNEG represents the OD values of the SPF cats and STD ODNEG indicates the standard deviation between the SPF cats’ OD values [The cutoff value indicating a positive reaction was determined using the equationD values .t-test (IBM SPSS©) assuming equal variance via Levene’s test, where p-values above 0.05 were considered not significant.Samples with OD readings between 1 and 2× the cutoff value were considered to be in the indeterminate range. OD values between the sample groups were compared using independent E. coli antibody. The FIV ELISAs were run as described in the ELISA protocol, only modifying which blocking reagent was utilized. The BSA, nonfat milk, and casein were tested using 3 SPF FPV+ cat serum samples. The anti-E. coli antibody was tested using 4 field cat serum samples.In order to reduce nonspecific binding, different blocking reagents were tested for the FIV ELISA. The reagents that were utilized were 2% BSA, 5% nonfat milk, 1% casein, and 1:200 anti-


Tandem repeat (TR) expansion is the underlying cause of over 40 neurological disorders. Long-read sequencing offers an exciting avenue over conventional technologies for detecting TR expansions. Here, we present Straglr, a robust software tool for both targeted genotyping and novel expansion detection from long-read alignments. We benchmark Straglr using various simulations, targeted genotyping data of cell lines carrying expansions of known diseases, and whole genome sequencing data with chromosome-scale assembly. Our results suggest that Straglr may be useful for investigating disease-associated TR expansions using long-read sequencing.The online version contains supplementary material available at 10.1186/s13059-021-02447-3. Long-read or third-generation DNA sequencing is increasingly employed in genomic applications such as de novo assembly, structural variant detection, transcriptome profiling, and metagenomics . The OxfShort tandem repeats (STRs) are genomic segments typically composed of 2–6 base pair (bp) units repeated in tandem and in the same orientation. STRs comprise about 3% of the human genome and are highly polymorphic. Lengths of certain STRs play biological roles. There are human diseases caused by STR expansions, where STR lengths beyond a certain threshold are known to be pathogenic . AdditioConventionally, clinical STR genotyping is performed by polymerase chain reaction (PCR) or Southern blot assays . Both meLong-read sequencing offers an opportunity to remedy these problems, especially for STRs that are kb, and sometime tens of kb, in length . LibraryMore than 40 diseases have been found to be caused by STR expansions . Repeat We developed Straglr, a new software tool that scans the entire genome for potential TR expansions by first extracting insertions composed of TRs and then genotyping the identified “expanded” loci. This approach not only spares the time and computing resources required for genotyping thousands of non-expanded TR loci but also enables the discovery of expansions at previously unannotated loci. Herein we have demonstrated Straglr’s performance in genotyping STR expansions using both simulated and real long-read data and evaluated its results of a genome scan against a diploid genome assembly.From the human reference genome (hg38), we generated three genomic sequences, each modified at 17 known STR disease loci , whereas tandem-genotypes showed p values smaller than the cutoff in two samples (expansions 100 and 500 bp). RepeatHMM exhibited a statistically different (p < 0.01) repeat size distribution from the actual one at all expansions, mostly likely due to its under-sizing of the larger alleles, which was visually observable in all cases.To evaluate the performance of each tool in genotyping STRs from long reads, we first compared repeat size distributions determined from individual reads against the actual size distributions Fig. a. To detWe then compared the numbers and identities of target-repeat harboring reads reported by the three tools against the ground truth Fig. b. As RepATXN1 in base size 1000 bp, separation 100 bp). We further extended the study by adding an additional allele with twice the separation size in each sample to simulate tri-allelic loci. Similar results to the bi-allelic samples were observed for Straglr  samples that contained different relative abundances of a premutation  and full mutation  HTT CAG expansions, three samples with FMR1 CGG expansions, and one negative control without any known repeat expansions. The data were generated by PacBio’s No-Amp targeted sequencing, an application that combines target enrichment using the CRISPR/Cas9 system with SMRT sequencing [HTT, FMR1, ATXN10, and C9orf72. The latter two known disease loci were included in PacBio’s analysis as negative controls. We compared the repeat size distribution on each locus extracted from Straglr’s results against those determined by PacBio’s analysis for each sample  sequences (SRR7615963) and high-quality diploid assembly GCA_003634875.1)  from HG034875.1  n = 215,894) that all three tools were able to genotype. We plotted the mean repeat sizes determined from individual support reads against the average repeat sizes determined from each assembly scaffold for each locus. Higher Pearson correlation coefficients for Straglr and RepeatHMM (R = 0.99) demonstrated these tools sized TRs more accurately than tandem-genotypes (R = 0.98) . Straglr’s genotyping results . We plotted the TR sizes reported by Straglr versus those determined from the assembly. The mean of the two allele sizes was used for plotting in Fig. R = 0.99) between the allele sizes called by Straglr and the assembly was observed . Of these, we observed 315 (72%) where both alleles agreed with the assembly, 106 (24%) agreed with one allele, and 16 (4%) agreed with neither , which also occurs at a short (six copies) GAA tract that is not included in the UCSC simple repeat annotations.Of the 6936 loci with alleles called as “expanded” by Straglr, 146 were not annotated as simple repeats by UCSC. At one of these loci at chr9:127,882,557-127,882,603, Straglr found a stretch of nine to 10 impure GAA repeats in the reference sequence that was “expanded” to over 140 copies in HG00733. This result was confirmed by checking the corresponding locus in the assembly manually  at different read depths: 11, 16, 22, 33, 44, and 87X  to study the effect of coverage depths on runtime of the software. To provide an idea of running the entire pipeline, we also included the runtimes of minimap2 alone and the sum of the runtimes of minimap2 and Straglr in the benchmarking Fig. ” sectionWe designed different simulation experiments to evaluate Straglr’s genotyping performance. We first demonstrated Straglr’s superior specificity and sensitivity see the “” sectionStraglr was developed around genome alignments produced by minimap2  because NOP56 of spinocerebellar ataxia 36.One challenge of detecting TRs in noisy long reads is that interruptions introduced by indels and base substitutions make the task of motif recognition difficult. Running tandem repeats finder (TRF) with lenient parameters in Straglr enabled TRs with imperfect or complex motifs to be detected. Nonetheless, occasional failures may occur in short alleles where deletions can cause disruptions big enough to preclude motif recognition, an example being the normal allele of 42 bp GGCCTG in Another challenging task during the genotyping phase is comparison of TR motifs identified from individual locus-spanning reads against the target motif, either provided by the user or detected by Straglr during the genome-scan phase. This is particularly difficult with VNTRs as longer TR sequences are inherently more error-prone and divergent. We employed BLASTN with reduced seed length to perform matching between long TR motifs, and imperfect identity with target motifs was accepted as positive matches. This motif screening procedure added extra running time for Straglr but was necessary to avoid STR genotyping mistakes. We provide an example at chr8:23,627,962-23,628,004 in HG00733 where an insertion of ~ 3.5 kb was observed in 35 out of 86 reads  between the sizes of annotated TRs determined by Straglr and the corresponding loci from the assembly  was observed between TR sizes from homozygous loci detected by Straglr’s genome scan and their corresponding sizes in the assembly. We observed a lower level percentage (73%) of heterozygous loci where both allele sizes agreed between Straglr and the assembly. As reported in the “We performed a genome scan on the entire SRR7615963 read set from HG00733 to search for TR expansions at least 100 bp larger than the allele in the reference genome. We conducted evaluations of identified homozygous and heterozygous “expanded” loci separately because the latter requires comparisons at both alleles. A high degree of correlation  of reference genomic sequence immediately up- or downstream of the position where the possible sub-sequence lies to the clipped read sequence using BLASTN. A successful unambiguous alignment provides the missing boundary of the repeat sub-sequence with the read and enables Straglr to extract the sequence for TRF inspection. TRF is again used to screen all candidate repeat sequences extracted, as motifs detected are matched against the target motif. When simple string matching using Python’s regular expression fails, such as the cases for long and complicated VNTRs, BLASTN is performed with mismatch allowance to discern matches between target and detected motifs. If none of the detected motifs matches the target motif, no genotyping result will be produced for the locus in question. The final size and motif sequence for each supporting read are extrapolated from TRF results. This process captures potential TRs smaller than the alleles identified from genome-scan mode (if genome-scan mode was run), hence compiling a complete list of alleles for genotype ascertainment.Straglr uses GMM (Python scikit-learn package) to estimate the genotype given all the repeat sizes identified at each target locus. It attempts different numbers of clusters up to a user-specified maximum (default = 2) and assigns the one with the smallest Akaike information criterion (AIC) value as the number of alleles. The median of all repeat sizes within each cluster is the size reported for that allele. The final output of Straglr details the supporting read names together with the copy numbers, sizes, and start location of the TR detected in each read.To simulate TR expansions, we replaced specific TRs Table  in the rTo identify reads that spanned the targeted loci modified with expansions, the origin of each simulated read was first deduced from NanoSim’s read names: in the underscore-separated fields of the read name, the first field represents the chromosome name, the second field represents the start genomic coordinate, the third field indicates whether the read is mappable to the genome  or not , and the seventh field represents the alignment length of the read, which by adding to the start coordinate yields the end genomic coordinate of the read. The coordinates of all “aligned” reads were intersected with the target locus coordinates with expansion sizes added to the ends, and the subsets that overlapped by at least 50 bp on both sides of the expansions were treated as the set of ground truth reads that harbor the expansions.FMR1 repeat locus  with 150 CGG repeats (PM) and the other with 500 repeats (FM), each with 500 kb reference sequences flanking the locus. We then generated 2000 simulated Nanopore reads by running NanoSim on each modified sequence, aiming to result in approximately 30 reads covering the locus. This step was repeated ten times to generate inputs for each mosaic composition. We then aligned all simulated reads against hg38 with minimap2 and identified all PM and FM reads that flanked the FMR1 repeat locus by at least one kb on both sides. From these sets, reads were randomly picked to make up the desired composition for each sample with a target combined coverage of 30X. Starting with samples composed of 100% PM reads, we produced a series of samples by serially shifting 10% of the input reads from PM to FM until the final samples that were composed of 100% FM reads. Each sample was aligned with minimap2 and genotyped by Straglr. The genotype output columns, with both the copy numbers (numbers of repeat units) detected and the number of supporting reads associated with each allele, were used as the inputs for generating Fig. We generated two modified genome sequences, one replacing the https://downloads.pacbcloud.com/public/dataset/RepeatExpansionDisorders_NoAmp/. In the “analysis” sub-directory, BAM files used for Straglr analysis resided under “align,” and per-read repeat sizes were extracted from the “totalLength” columns of the “*.counts.tsv” files under “reports.” Per-read repeat sizes determined by Straglr were taken from the “size” field of its outputs. Target repeat coordinates and motifs used for running Straglr in genotype-only mode were copied from the “human_hs37d5.targets_repeatonly.bed” file under the “auxillary” sub-directory.Alignments and analysis results were downloaded from PacBio’s No-Amp Targeted Sequencing dataset repository: To determine TR sizes from the assembly, we extracted 500 bp reference genome sequences flanking either side of the TR coordinates (left and right probes) and aligned them against the assembly with BWA mem . Only unTo determine if heterozygous alleles of a given TR locus ascertained by either Straglr or RepeatHMM agreed with the corresponding alleles from the diploid HG00733 assembly, we sorted the allele sizes and compared the smaller and larger alleles from each source independently. A match was declared when both alleles were within 20 bp in size or the difference was within 10% of the assembly allele size. True positives were represented by heterozygous genotype calls in which both alleles matched the assembly using the above criteria, with the total number of heterozygous calls or the total number of heterozygous alleles identified in the assembly as the denominator in the calculation for sensitivity and specificity respectively.Additional file 1. Supplementary Figs. S1-S6 and Table S1. Supplementary benchmarking results.Additional file 2. Supplementary Table S2. Software versions and parameters used in all analysis in this manuscript.Additional file 3. Size estimates of target loci from both PacBio proprietary analysis and Straglr for all sequencing reads in No-Amp targeted sequencing data. (TSV 862 kb)Additional file 4. Review history.


Aims: The aims of the current study were as follows: 1) to assess gerontology graduate students’ beliefs about medical marijuana’s (MMJ) effectiveness for two common age-related conditions - Alzheimer’s (AD) and Parkinson’s disease (PD); 2) to assess students’ beliefs and attitudes toward MMJ; 3) to explore associations linking background characteristics, MMJ-related attitudes and beliefs, and beliefs about the MMJ effectiveness for AD and PD. Method: A sample of 104 (84 women and 20 men) gerontology graduate students voluntarily participated in an anonymous online survey. Results: The vast majority (95%) of the participants indicated they had no formal education about MMJ and reported being unprepared to answer clients’ MMJ-related questions (84.6%). Most of the participants believed that MMJ is effective for use with AD (70.2%) and PD (80.8%) patients. Participants reported favorable beliefs about MMJ benefits, concerns about risks, the need for training, and positive attitudes toward recreational marijuana use legalization. Prior marijuana use  was found to be associated with more positive beliefs about MMJ benefits, risks, and its legalization for recreational purposes. Prior marijuana use was the only factor associated with the belief that MMJ is an effective therapy for use with individuals diagnosed with AD or PD. Conclusions: The study findings stress the need for students’ MMJ education in order to provide future gerontology service providers with the necessary knowledge and ability to address clients’ questions about MMJ use. Efforts to develop curricula and training programs need to be promoted.


OPN1LW and OPN1MW. There was also benefit in investigation of the repetitive GC-rich ORF15 region of RPGR. Further molecular investigations were facilitated by focused assays in these regions. Deep intronic variants were identified in IQCB1 and ABCA4, with functional RNA based studies of the IQCB1 variant revealing activation of a cryptic splice acceptor site. While targeted capture panel-based methods are successful in achieving an efficient molecular diagnosis in a proportion of cases, this study highlights the additional benefit and clinical value that may be derived from WGS, focused assays and functional genomics in the highly heterogeneous IRDs.The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy. In this study, we investigated whole genome sequencing (WGS), focused assays and functional studies to assist with unsolved IRD cases and facilitate integration of these approaches to a broad molecular diagnostic clinical service. The WGS approach identified variants not covered or underinvestigated by targeted capture panel-based clinical testing strategies in six families. This included structural variants, with notable benefit of the WGS approach in repetitive regions demonstrated by a family with a hybrid gene and hemizygous missense variant involving the opsin genes,  Further complexities exist with single genes contributing to several types of IRD or variant types in a gene which may lead to disease in autosomal dominant or autosomal recessive forms, such as PRPH2 [OMIM: 179605] and PROM1 [OMIM: 604365]. These factors all lead to challenges in obtaining an accurate genetic diagnosis, which is essential for access to emerging genetic therapies which require a specific genetic diagnosis for clinical trial or therapy eligibility  (two families), and other copy number variants (SV/CNVs) (two families).RPGR was interrogated with Sanger sequencing with no likely causative variant identified.In Family 1, a large non-consanguineous family of middle eastern ethnicity presented with nystagmus for ophthalmic and clinical genomic review. Initial clinical ophthalmic examination of the proband A–D reveaOPN1LW and OPN1MW opsin gene read data in IGV took place and was suggestive of a structural variant, with a reduction in the read depth when available data from the two affected males in the family were compared to unaffected male WGS data of the same loci (OPN1MW(NM_000513.2):c.607T>C p.(Cys203Arg) was also detected in all available reads, which has been reported in association with a single 5′ OPN1LW, 3′ OPN1MW hybrid gene chr9:2668923_2770120del , which is an autosomal recessive disorder  and a reverse primer specific to exon 4 of OPN1MW coding sequences [RVS: 5′-CACGATGATGCTGAGTGGGG-3′]. The presence of an amplifiable PCR product using these primers allowed for confirmation of the hybrid opsin gene and also for the detection of obligate female carriers.Primers were designed to confirm the hybrid opsin gene for this particular family, with a forward primer specific to exon 3 of ® SV Gel and PCR Clean-Up System  before Sanger sequencing .Fibroblasts were cultured from a skin biopsy from the proband from Family 3 and total RNA was extracted using the RNeasy micro kit . Total RNA was converted to cDNA using the SuperScript IV 1st Strand Synthesis System kit . Primers were designed using Primer3, with forward primers located in exons 3 and 4, with reverse primers located within exons 5 and 6, and sequences are available on request. PCR was performed following standard cycling conditions with amplified products resolved on agarose gel electrophoresis. Both wild type and mutant bands were excised and purified using the WizardWhile targeted capture panel-based methods are a widely applied and successful strategy in achieving an efficient molecular diagnosis in a proportion of IRD cases, this study has highlighted WGS as a superior technology for detection of variants in repetitive and/or GC rich regions, deep intronic regions or where there are small or complex SVs/CNVs, which together were implicated in 6% of a heterogenous IRD cohort. However, detection of these variants often requires focused, detailed analysis of the WGS data informed by further clinical phenotype or pedigree information. In addition, focused assays, orthogonal approaches and functional studies are frequently required to validate the identified variants. These factors emphasize the need for development of efficient approaches for incorporation of genomic sequence data analyses and functional genomics approaches into the clinical diagnostic workflow.


Post-translational acylation of lysine side chains is a common mechanism of protein regulation. Modification by long-chain fatty acyl groups is an understudied form of lysine acylation that has gained increasing attention recently due to the characterization of enzymes that catalyze the addition and removal this modification. In this review we summarize what has been learned about lysine fatty acylation in the approximately 30 years since its initial discovery. We report on what is known about the enzymes that regulate lysine fatty acylation and their physiological functions, including tumorigenesis and bacterial pathogenesis. We also cover the effect of lysine fatty acylation on reported substrates. Generally, lysine fatty acylation increases the affinity of proteins for specific cellular membranes, but the physiological outcome depends greatly on the molecular context. Finally, we will go over the experimental tools that have been used to study lysine fatty acylation. While much has been learned about lysine fatty acylation since its initial discovery, the full scope of its biological function has yet to be realized. Post-translational modification of proteins is a key biological regulatory mechanism that impacts every aspect of life. One class of post-translational modifications is protein lipidation which involves the attachment of hydrophobic moieties such as fatty acyl groups, isoprenoid lipids, or cholesterol . These h14) and palmitoylation (C16) are the most common forms of KFA, but the identity of the endogenous acyl group is often unknown making fatty acylation a more inclusive and general description (Lysine fatty acylation (KFA) is the addition of long-chain fatty acyl groups to lysine side chains via amide bonds. Myristoylation (Ccription . KFA of 2+-dependent histone deacetylases (HDACs) and the NAD+-dependent sirtuins (SIRTs). Archaebacterial sirtuins Sir2Af1 and Sir2Af2 from the archaea Archaeoglobus fulgidus, were also reported to be able to remove KFA, but there are no known substrates for this activity  family of proteins. This includes both Znactivity . The KFAactivity . Sir2A fentified . Human ein vitro . We willin vitro SIRT6 deacetylase activity is relatively weak compared to other sirtuin family members, raising the possibility of additional enzymatic activities  toxins. RTX toxins function in various ways, but typically act as cytolysins by forming pores in the plasma membrane of mammalian cells . Structu by RtxC .Escherichia coli hemolysin toxin HlyA. Initial characterization of the HlyA toxin revealed that HlyC activates proHlyA in a manner dependent on the acylated acyl carrier protein (acyl-ACP)  . Follow-cyl-ACP) . While tcyl-ACP) . Similare lysine . Interesd lysine . This reMore detailed information of RtxC enzymology was revealed using HlyC . HlyC acActinobacillus pleuropneumoniae, has conspicuous structural homology to the Gcn5-like N-acetyl transferase (GNAT) superfamily  toxins. MARTX toxins contain the trademark non-apeptide repeats in both the N- and C-terminus, which forms a pore in the cell membrane that translocates the interior portion of the toxin encoding a modular array of effector domains into the cytoplasm . While tVibrio cholerae toxin, MARTXVc (Vc is known to induce cell rounding through an actin crosslinking domain (ACD) that covalently links actin monomers, preventing actin polymerization  domain, function to suppress proinflammatory signaling. Cytoskeletal collapse caused by ACD activates mitogen-activated protein kinase (MAPK) signaling, leading to upregulation and enhanced secretion of proinflammatory cytokines. RID inactivation of Rac1 blocks MAPK signaling and subsequent cytokine secretion  have long been known to transfer a myristoyl group from myrsitoyl-CoA to the α-amine of an N-terminal glycine following cleavage of the initiator methionine . This moBecause NMTs catalyze the fatty acylation of a primary amine, they can do the same chemistry as a potential KFA transferase. Indeed, two groups found that the human NMT1 and NMT2 can catalyze KFA on lysines toward the N-terminal of a peptide . The proA diverse set of proteins has been identified to have KFA . How exaInterleukins (ILs) are a family of secreted proteins used in cell-to-cell communication that regulate inflammatory signaling. The IL-1 family of proteins contains 11 different members that are synthesized as precursor proteins, requiring proteolytic processing for optimal biologic activity . IL-1α aAquaporins are a family of transmembrane channel proteins that facilitate the transport of water across the plasma membrane. Aquaporin-0 (AQP0) is highly abundant in the ocular lens where it plays an important role, not only in circulating water, but as a cell to cell adhesion protein for maintenance of proper tissue structure . There iTumor necrosis factor α (TNF-α) is a proinflammatory cytokine secreted by several immune cells. TNF-α is translated with a single transmembrane domain that is cleaved at the plasma membrane to release a soluble form that binds TNF receptors (TNFRs). TNFR1 is universally expressed on almost every cell type meaning that TNF-α signaling is both ubiquitous and tightly regulated . In a foThe only other known endogenous substrate for SIRT6 deKFA activity is R-Ras2, a member of the Ras family of GTPases . Ras GTPKRAS  is the most frequently mutated gene in cancer, so identifying how modifications regulate its activity is of great interest  subfamily that has been found to be modified with KFA . While min vitro activity in converting serine and THF to glycine and methylene-THF. Instead, KFA was found to be relevant in SHMT2’s role in the BRISC complex. Increasing SHMT2 KFA, through HDAC11 knock out or knock down, increased SHMT2 localization to the endosomes/lysosomes where the BRISC complex can hydrolyze IFNαR1 ubiquitination. Correspondingly, HDAC11 KO cells had more IFNαR1 on the cell surface following IFNα treatment. Further, HDAC11 KO cells had stronger downstream signaling following IFNα stimulation and HDAC11 KO mice also had a stronger antiviral response when challenged with vesicular stomatitis virus. In this context, KFA again controls intracellular localization of the substrate protein, here leading to enhanced cellular activity of a deubiquitylase complex. Inhibiting HDAC11 may therefor enhance antiviral responses.Serine hydroxymethyltransferase 2 (SHMT2) is a key member of one-carbon metabolism. SHMT2 catalyzes the reversible conversion of serine and THF to glycine and methylene-THF in the mitochondria. High levels of SHMT2 are associated with poor patient prognosis in several cancers and inhibitors that target both SHMT2 and SHMT1  have shown promising results in initial cellular and mouse studies . AdditioE. coli and B. pertussis, HlyA and CyaA, respectively, are both able to form pores in liposomes and planar lipid bilayers  . In thisIf a target protein can be readily purified in large quantities, endogenous KFA levels can be analyzed with mass spectrometry, such as for APQ0 . However3H- and 14C-labeled fatty acids are the best choice for this application as the isotopes can be incorporated into the fatty acid without modifying the structure. However, these isotopes have relatively low signal and must be monitored for a long time.One class of exogenous fatty acyl probes is radioactive-isotope-labeled fatty acids. Cells treated with radioactive fatty acids can then form radioactive fatty acyl-CoA for use by KFA transferases. A protein of interest can then be purified and monitored for radioactivity to assay for fatty acylation . 3H- andClickable fatty acid analogs have proven to be a more powerful tool for studying KFA. After treating cells with these probes, proteins are modified with fluorophores or biotin via click chemistry to track levels of fatty acylation using fluorescence scanning, western blot, or various mass spectrometry approaches . While aSince the initial discovery of KFA on IL-1α, significant progress has been made in studying KFA. This is especially true of the last 10 or so years as several enzymes were identified to add or remove KFA. Additionally, implementation of clickable probes has allowed for the identification of many new proteins with KFA and has hastened the pace of discovery. We now know that KFA can regulate a variety of biological processes such as protein secretion, tumorigenesis, and immune signaling. The knowledge gained surrounding KFA can now serve as a basis for further exploration.Multiple species of pathogenic bacteria utilize KFA to enhance their pathogenesis. While mammalian cells are not yet known to have KFA transferases that act on a broad range of substrates, the presence of multiple enzymes with strong KFA hydrolase activity suggests an evolutionarily beneficial role for this activity. Is it possible that during certain bacterial infections KFA hydrolase activity serves a protective role against toxins like RID and IcsB? Is it possible that KFA hydrolase enzymes could remove RTX toxin KFA to inactivate the pore-forming toxin?Additional lines of future study are myriad. In addition to bacteria, could other pathogens modulate KFA during infection? In addition to infection, KFA has already been shown to modulate tumor phenotypes. What other diseases could be regulated by KFA? Can KFA guide the creation of therapeutics to counteract these diseases? Addressing these questions, will require identifying more proteins modified by KFA and additional enzymes that regulate this modification, as well as the development of small molecules targeting the regulatory enzymes. Future work needs to address these issues to fully understand the biological impact of KFA.GK and HL surveyed the literature and wrote the manuscript. Both authors contributed to the article and approved the submitted version.HL was a founder and consultant for Sedec Therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Casp8IECΔ mice). Deletion of STAT1 in Casp8IECΔ mice was associated with reduced cell death and a partial rescue of Paneth cell function in the small intestine. Likewise, organoids derived from the small intestine of these mice were less sensitive to cell death triggered by IBD-key cytokines such as TNFα or IFNs. Further functional in vitro and in vivo analyses revealed the impairment of MLKL-mediated necrosis as a result of deficient STAT1 function, which was in turn associated with improved cell survival. However, a decrease in inflammatory cell death was still associated with mild inflammation in the small intestine. The impact of STAT1 signaling on gastrointestinal inflammation dependent on the localization of inflammation, as STAT1 is essential for intestinal epithelial cell death regulation in the small intestine, whereas it is not the key factor for intestinal epithelial cell death in the context of colitis. Of note, additional deletion of STAT2 was not sufficient to restore Paneth cell function but strongly ameliorated ileitis. In summary, we provide here compelling molecular evidence that STAT1 and STAT2, both contribute to intestinal homeostasis, but have non-redundant functions. Our results further demonstrate that STATs individually affect the distinct pathophysiology of inflammation in the ileum and colon, respectively, which might explain the diverse outcome of JAK inhibitors on inflammatory bowel diseases.Blocking interferon-function by therapeutic intervention of the JAK-STAT-axis is a novel promising treatment option for inflammatory bowel disease (IBD). Although JAK inhibitors have proven efficacy in patients with active ulcerative colitis (UC), they failed to induce clinical remission in patients with Crohn's disease (CD). This finding strongly implicates a differential contribution of JAK signaling in both entities. Here, we dissected the contribution of different STAT members downstream of JAK to inflammation and barrier dysfunction in a mouse model of Crohn's disease like ileitis and colitis ( Caspase-8 as well as Mlkl gene expression in intestinal epithelial cells (IECs) to induce apoptosis and necroptosis  are grouped into three families: Type I with IFNα, IFNβ, and several minor subtypes; Type II with IFNγ and Type III with IFNλs. Their expression can be induced in response to diverse viral and bacterial stimuli in an autocrine or paracrine fashion –3. Ligatroptosis . In controptosis .Casp8IECΔ) in intestinal epithelial cells spontaneously develop intestinal inflammation with histomorphological alterations similar to the classical features of Crohn's disease. Accordingly, Casp8IECΔ mice mimic several important characteristics including Paneth cell depletion accompanied by microbial dysbiosis, the culprit of inflammation in the ileum, as well as the immune cell signature (Th1 driven) association with elevated IFN levels  in the drinking water for 5 days. The development of colitis was monitored with a high-resolution video mini-endoscopic system. Endoscopic scores for intestinal inflammation were assigned based on the criteria described for the assessment of the murine endoscopic index of colitis severity, which scores the following parameters: translucency, granularity, fibrin, vascularity, and stool consistency, as previously described .Tissue for histology and immunohistochemistry were collected and fixed in 4.5% PFA. Tissue was embedded in paraffin in a water-free procedure and stored at room temperature for further analysis. Samples for RNA and protein analyses were instantly frozen in liquid nitrogen and stored at −80 °C until further use.In-Situ Cell Death Detection Kit (Roche). Images were obtained using the microscope LEICA DMI 4000B together with the LEICA DFC360 FX or LEICA DFC420 C camera or the microscope Leica DMi1 with the corresponding imaging software.Histopathological analyses were performed on formalin-fixed paraffin-embedded tissue cross sections after Mayer's haematoxylin and eosin (H&E) staining or a combined staining with periodic acid–Schiff reaction and alcian blue (PAS). Immunofluorescence of tissue sections was performed using Streptavidin Protein DyLight (Thermo). Primary antibodies (for detailed information see hypoxanthine guanine phosphoribosyl transferase (Hprt) or Glyceraldehyde 3-phosphate dehydrogenase (Gapdh). For fold change calculation, the average mean of the relative expression of control mice were set as 1.Total RNA was extracted from intestinal tissue using the peqGOLD Total RNA Kit or Total peqGOLD Microspin Total RNA Kit for organoids (peqLab/VWR). cDNA was synthesized by reverse transcription using the SCRIPT cDNA Synthesis Kit (Jena Bioscience) and analyzed by real-time qPCR using SYBRGreen reagent (Roche), the LightCycler 480 (Roche) and specific QuantiTect Primer Assays (Qiagen)  for a minimum of 7 days according to Sato et al. . Organoit-test. Comparisons among multiple groups were performed using ANOVA followed by multiple comparison and statistical significance was accepted with p < 0.05 . Statistical calculations were performed using GraphPad Prism 8 (GraphPad Software).Comparisons of two groups were performed using an unpaired two-tailed STAT1 signaling attenuates MLKL mediated necroptosis in the ileum and partially restores Paneth cell function.Mlkl gene transcription via activation of the transcription factor STAT1 in hepatocytes . Generation of double deficient mice strain (Casp8IECΔxStat1−/−) was not trivial, as both genes are closely located on chromosome 1 , suggesting that STAT1 might be relevant upstream of Caspase-8.Previously, we have demonstrated that IFNs can trigger programmed necrosis by regulating atocytes . Further ileitis . To studCasp8IECΔ mice are characterized by MLKL mediated epithelial necroptosis, Paneth cell depletion and ileitis (Mlkl in Casp8IECΔ mice (Casp8IECΔxMlkl−/− mice) was sufficient to block Paneth cell death and inflammation  positive dying cells   developed equal colonic inflammation  . Hence,  colitis . Casp8ΔIstration . Interes−/− mice , while eammation . Furtherammation . In conttermates . These rCaspase-8 and Stat2 double deficient mice (Casp8IECΔx Stat1+/−Stat2−/− mice) as well as triple knockout animals lacking both STAT1 and STAT2 members (Casp8IECΔx Stat1−/−xStat2−/−). Similar to organoids derived from Casp8IECΔxStat1−/− mice, Paneth cell numbers and expression of antimicrobial peptides was not influenced by additional deletion of Stat2 in Casp8IECΔ mice in vitro   . In lineIEC mice . In contIEC mice , suggestCasp8IECΔ mice display severe inflammation including bowel wall thickening and an increased cellularity in the lamina propria of the terminal ileum. By contrast, deletion of STAT2 in these mice decreased these histo-morphological features  as potential novel player in the pathogenesis of intestinal inflammation. ZBP1, also known as DAI, was initially identified to induce IFN-mediated MLKL-dependent necroptosis in the context of viral infection (Here, we provided novel evidence that STAT1 and STAT2 might independently influence intestinal inflammation in a highly spatial-dependent process. Blocking STAT1 signaling by genetic deletion of this transcription factor in ithelium . These dithelium . Accordiithelium , 36. Morithelium . Here, wnfection , 38. Hownfection , 39. Intin vitro and partially rescued Paneth cell death in vivo, it was surprisingly not involved in inflammation in the small intestine. Moreover, we identified that STAT1 signaling might be associated with tissue injury processes in the colon as Casp8IECΔx Stat1−/− mice exhibited severe tissue injury and inflammation in response to experimental colitis. These data are in line with a previous publication by Chiriac et al., highlighting that the activation of IFNλ-STAT1 signaling specifically in the IECs is responsible for mucosal healing and epithelial regeneration during colitis (Casp8IECΔ mice, was associated with mucosal healing and reduction of disease activity in the small intestine, while Paneth cell homeostasis was not influenced by STAT2. STAT2 is linked to type I interferon signaling and only little is known about its role during IBD (Beside their impact on cell death regulation, IFNs are primarily known for their immune-modulatory function. Accordingly, while STAT1 was sufficient to block TNFα or IFN triggered cell death  colitis . Our resring IBD . In humaring IBD , 40. Furin vitro and in vivo (Our findings on the differential role of STAT signaling molecules in the context on ileitis and colitis are in line with previous studies, supporting the concept that the pathogenic mechanism underlying ileal and colonic Crohn's disease are distinct and thus require individual therapies. Our data now provide further mechanistic insights in the role and contribution of the JAK-STAT signaling in the intestinal tract, which is currently in clinical focus. Accordingly, blocking JAK-STAT signaling is a promising therapeutic intervention, but current studies uncovered differential therapeutic success between ulcerative colitis and Crohn's disease –26. The  in vivo . In lineIn summary, we provide molecular evidence that STAT1 and STAT2 both contribute to intestinal inflammation but have differential functions. Our results demonstrate that STAT1 coordinates cell death in the ileum but not during experimental colitis. Furthermore, STAT2 was able to modulate mucosal inflammation, independent of STAT1. Thus, our data provide further evidence for a differential pathological mechanism responsible for ileal and colonic inflammation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Regierung von Unterfranken and conducted by qualified personnel.IS, MN, and CG: designed the research. IS and AD: performed the experiments. MC: supplied material. IS, MN, and CG: analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila between flies reared on 12:12 and 8:8 (simulated shift work) light:dark schedules. Actimetric analysis showed that the 8:8 light:dark schedule effectively disrupted the rhythms in locomotor output. Inaccurate NHEJ repair was not a frequent outcome in this system overall, and no significant difference was seen in the usage of NHEJ or SSA repair between the control and simulated shift work schedules. We conclude that this circadian disruption regimen does not alter the usage of mutagenic NHEJ DSB repair in the Drosophila male pre-meiotic germline, in the context of the Rr3 system.Long-term shift work is widely believed to increase the risk of certain cancers, but conflicting findings between studies render this association unclear. Evidence of interplay between the circadian clock, cell cycle regulation, and DNA damage detection machinery suggests the possibility that circadian rhythm disruption consequent to shift work could alter the DNA double-strand break (DSB) repair pathway usage to favor mutagenic non-homologous end-joining (NHEJ) repair. To test this hypothesis, we compared relative usage of NHEJ and single-strand annealing (SSA) repair of a complementary ended chromosomal double-stranded break using the Repair Reporter 3 (Rr3) system in  Engagement in shift work that involves light during subjective night has been identified as a probable carcinogen . Long-teThe most immediate effect of night-shift work is the disruption of the circadian rhythm by exposure to light during subjective night, inconsistent sleep schedules, and irregular mealtimes . The possibility of a mechanistic link between circadian rhythm disruption and tumor initiation is less well examined. The key event in tumor initiation is thought to be the accumulation of mutations via unrepaired or misrepaired DNA damage (reviewed in ). Some eA connection between the circadian clock and the cellular response to DSBs is especially salient because DSBs are a particularly genotoxic lesion. Since both strands are compromised, the potential for mutation is high. Genetic disorders that impair the ability to detect or repair DSBs are typically cancer prone syndromes (reviewed in ). DSBs cRepair pathway choice depends on a number of factors, including capacity to detect the break and cell cycle phase . EvidencSceI endonuclease. Repair of the endonuclease-induced break by SSA activates DsRed expression, and repair by NHEJ does not. Therefore, the repair mechanism can be identified by a simple visual screen. Flies containing both Rr3 and an I-SceI expression construct are generated by appropriate crosses. Breakage and repair events take place in the male pre-meiotic germline, and are recovered by appropriate crosses to be scored in the next generation.To test the hypothesis that circadian rhythm disruption can alter DNA DSB repair pathway choice, we used the Repair Reporter 3 (Rr3) system  to examiWe found that the 8:8 L:D schedule effectively abrogated normal activity rhythms. No difference was seen in the relative use of SSA and NHEJ between flies kept on the 12:12 and 8:8 schedules. We conclude that in the Rr3 system, the choice of NHEJ versus SSA repair is not affected by this method of circadian rhythm disruption.1118Fly stocks and husbandry. w flies were the kind gift of F. Rob Jackson (Tufts University). Rr3 and P{UIE} flies [E} flies  were theActivity monitoring. Time courses of locomotor activity were recorded using the Drosophila Activity Monitor (DAM) system  [https://karolcichewicz.shinyapps.io/shinyr-dam/ (accessed on 8 January 2022). Circadian period length was analyzed by the Chi-square periodogram [tham MA) . DAM5M mtham MA) , using tiodogram  implemenRT-qPCR. P{UIE} flies (2–3 per sample) were collected under light ether anesthesia, immediately ground in Trizol LS reagent  and stored at −80°. RNA was extracted with chloroform and further purified with the RNA Clean and Concentrator–5 kit  with on-column DNase I digestion carried out according to the manufacturer’s instructions. 100 ng of RNA was used for reverse transcription with the iScript Advanced cDNA kit . qPCR was carried out according to standard methods in a BioRad CFX96 Touch 1000 thermocycler using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions with 0.5 µL of cDNA per reaction. Primers used to amplify the period gene were Per-F (5′–CAGCTGCAGCAACAGCCAGTCG–3′) and Per-R (5′–GGCCTGCGTCGAGGGCTTGC–3′). The ribosomal gene RpL32 was used as a constant expression control, with the primers RpL32-F (5′–GCCCAAGATCGTGAAGAAGC–3′) and RpL32-R (5′–CGACGCACTCTGTTGTCG–3′). Relative expression was calculated using the double delta Cq method [q method , using tThe Rr3 construct. The Rr3 DSB repair reporter construct [SceI endonuclease, flanked by a 147 bp repeat. The Rr3 construct additionally contains a functional copy of the white gene, which allows the presence of the Rr3 construct to be identified in the white mutant background via examination of eye color. When the Rr3 construct is cut by I-SceI, the resulting complementary ended DSB can be repaired by SSA, NHEJ, or HRR. SSA repair using the 147 bp repeat restores DsRed gene expression. Accurate NHEJ restores the original sequence, which can be cut again. Inaccurate NHEJ that alters the sequence of the I-SceI site prevents further DSB induction without restoring DsRed expression. HRR from an intact sister chromatid reconstitutes the original sequence, which can be cut again, and HRR from the homolog results in gene conversion and complete loss of the Rr3 site. All experiments used an Rr3 construct integrated into chromosome 2 at 48C. Full details of the construction of Rr3 are given in [onstruct  consistsgiven in .Induction of double-strand breaks and quantitation of repair events. When male flies harboring an Rr3 construct are crossed to female flies expressing the I-SceI endonuclease under a constitutive ubiquitin promoter (0 cross), F1 progeny undergo repeated cycles of DSB induction and repair in all cells. The timing of DSB induction depends on whether endonuclease is provided by maternal effect only, or by both maternal effect and zygotic expression of I-SceI. The chromosome containing the I-SceI expression construct contains a dominant mutant allele of the gene Stubble . In F1 progeny that inherit only the Rr3, DSB induction is due solely to maternal effect endonuclease in the egg, and continues until an inaccurate repair event takes place or maternal effect protein is exhausted . Relative usage of different DSB repair mechanisms in the F1 generation male pre-meiotic germline is quantitated by recovering individual repair events via appropriate crosses, and scoring F2 individuals for DsRed expression.bble Sb; , allowin1 germlines that have been exposed to both maternal effect and zygotically expressed endonuclease . To ensty peaks A. Chi-sqty peaks B. When tty peaks A and thety peaks C, consisty peaks .period gene, a key component of the negative feedback limb of the Drosophila circadian clock [period expression in P{UIE} flies showed the expected daily oscillations, albiet with a 4 h phase shift from the expected expression peak and nadir. In contrast, period expression in flies kept under 8:8 conditions did not show clear rhythmicity, and exhibited substantial inter-sample variability, suggesting that the 8:8 L:D schedule does disrupt the circadian clock at the molecular level.To assess the function of the circadian clock on the molecular level under 12:12 and 8:8 L:D conditions, we carried out an RT-qPCR expression time course of the an clock . ResultsWe wished to examine the effect of long-term circadian rhythm disruption on DNA double-strand break repair. However, in the Rr3 system with endonuclease expression controlled by a constitutive promoter, a substantial proportion of repair events occur early in the life cycle, before the effects of sustained circadian rhythm disruption can accumulate. To circumvent this technical limitation, we examined repair events mediated by maternal effect endonuclease in embryos produced by female flies that had been raised under either 12:12 or 8:8 conditions. We reasoned that before the onset of zygotic transcription, the repair capacity of the embryo would reflect that of the female parent.DsRed-expressing and -non-expressing flies were readily distinguished . ResultsDrosophila circadian clock begins to function during embryogenesis and can respond to light inputs during development [The elopment . TherefoHuman studies have produced conflicting results regarding whether long-term shift work should be considered a carcinogen. Resolution of this uncertainty will require elucidation of any mechanistic links between long-term shift work and initiation or promotion of tumorigenesis. Although potential links between circadian rhythm disruption and tumor promotion have been investigated in some detail, little previous work has directly examined the possibility of a link between circadian rhythm disruption and tumor initiation via increased usage of mutagenic DNA double-strand break repair.Previously published studies support the notion that susceptibility to DNA damage is time-of-day-dependent, and this dependence requires a functional circadian clock. For example, Plikus et al.  showed iTo investigate the possibility that circadian rhythm disruption could affect DNA double-strand break repair pathway choice, we compared relative usage of SSA and NHEJ in repairing a complementary ended DNA double-strand break between flies maintained on a conventional 12:12 light:dark schedule and a schedule designed to simulate rotating 8-hour shifts. Surprisingly, we observed no difference in usage of NHEJ repair between these conditions.Further investigation will be required to determine whether the results from this study can be generalized to other break and cell types. DSB repair pathway choice is strongly dependent on the sequence surrounding and structure of the break . The locebony gene, which has been shown to be involved in circadian locomotor output [ebony mutant flies have been shown to have normal circadian clock function [One additional consideration for interpreting results in the Rr3 system is a possible effect of the mutant alleles used as visible genetic markers. In particular, the P{UIE} flies are homozygous for a mutant allele of the r output . We do nfunction . HoweverDrosophila testis, and mutations in core clock genes impair male fertility [Period is expressed in the female Drosophila ovary [It is also possible that the cells comprising the embryonic or mature male pre-meiotic germline are not vulnerable to the deleterious effects of circadian rhythm disruption. We do note that core circadian clock genes are expressed in the ertility . Similarla ovary , althougla ovary . Alternala ovary ), which la ovary ). Thus,


SMAD4 or BMPR1A. The other half of JPS cases lack a known genetic driver  and are instead diagnosed clinically based on existing criteria including having five or greater pathologically defined juvenile polyps in the lower GI tract, juvenile polyps in the upper and lower GI tract, or any number of juvenile polyps with a family history of JPS. JPS is associated with increased risk of gastrointestinal cancer  . The. TheSMADSMAD4/BMPR1A. These differences are relevant for informing the clinical management of mutation-negative JPS patients, an area that previously lacked data to permit differing clinical recommendations. Consequently, our data were incorporated into the most recent National Comprehensive Cancer Network (NCCN) surveillance guidelines, which now separate mutation-negative JPS from JPS due to a SMAD4/BMPR1A mutation to allow for relaxed GI surveillance in mutation-negative JPS [SMAD4/BMPR1A variant [The differences observed in our multi-institutional JPS cohort show that mutation-negative JPS has a distinct phenotype compared to JPS with a mutation in tive JPS . Specifi variant . FurtherSMAD4 or BMPR1A.Additionally, the difference in heritability and polyp burden that our data highlighted may also suggest a difference in underlying disease pathogenesis in individuals without a detectable germline variant. For example, given the younger age of diagnosis and limited family history within this mutation-negative cohort, a lower-penetrance germline variant might be considered, or an autosomal recessive pattern of inheritance. Further, somatic mosaicism or epigenetic alterations might also drive the sporadic JPS phenotype. Further research is needed to better understand the cause of clinical JPS in patients who do not have a mutation in SMAD4 or BMPR1A have a distinct clinical phenotype that may potentially be viewed as a unique polyposis syndrome compared to JPS due to a SMAD4/BMPR1A variant. Although individuals with mutation-negative JPS still need to be followed closely, they may ultimately have lower overall cancer risk.In conclusion, our multi-center study is the largest study to date investigating the differences between individuals with or without a known genetic cause of JPS. Clear differences including younger age of presentation, lower chance of heritability, and lower burden of disease  all suggest that patients without a mutation in


Introduction: Bacterial biofilms are an important virulence factor inchronic periprosthetic joint infection (PJI) and other orthopedic infectionsince they are highly tolerant to antibiotics and host immunity. Antibioticsare mixed into carriers such as bone cement and calcium sulfate bone voidfillers to achieve sustained high concentrations of antibiotics required tomore effectively manage biofilm infections through local release. The effectof antibiotic diffusion from antibiotic-loaded calcium sulfate beads(ALCS-B) in combination with PMMA bone cement spacers on the spread andkilling of Pseudomonas aeruginosa Xen41 (PA-Xen41) biofilm was investigated using a “large agarplate” model scaled for clinical relevance. Methods: BioluminescentPA-Xen41 biofilms grown on discs of various orthopedic materials were placed within a large agar plate containing a PMMA full-size mock “spacer”unloaded or loaded with vancomycin and tobramycin, with or without ALCS-B.The amount of biofilm spread and log reduction on discs at varying distancesfrom the spacer was assessed by bioluminescent imaging and viable cellcounts. Results: For the unloaded spacer control, PA-Xen41 spread from thebiofilm to cover the entire plate. The loaded spacer generated a 3 cm zone ofinhibition and significantly reduced biofilm bacteria on the discsimmediately adjacent to the spacer but low or zero reductions on those further away. The combination of ALCS-B and a loaded PMMA spacer greatlyreduced bacterial spread and resulted in significantly greater biofilmreductions on discs at all distances from the spacer. Discussion: Theaddition of ALCS-B to an antibiotic-loaded spacer mimic increased the area of antibiotic coverage and efficacy against biofilm, suggesting that acombination of these depots may provide greater physical antibiotic coverageand more effective dead space management, particularly in zones where thespread of antibiotic is limited by diffusion (zones with little or no fluidmotion). While infrequent, PJI has a dramaticeffect on the patient's health, often resulting in joint dysfunction,morbidity, and mortality. Thefinancial burden placed on patients and the healthcare system is staggering. A major complicating factor in treatingPJI is microbiota-produced biofilms. Biofilms are pathogenic communities that adhere to living andnonliving surfaces and exhibit greatly increased antibiotic tolerance andresistance against host immunity. The establishment of biofilms is assistedby the presence of foreign materials of orthopedic implant components, suchas various metals and polymers.Pseudomonas aeruginosa is a Gram-negative opportunistic nosocomial pathogen and is cultured up to20 % of the time in chronic Gram-negative PJI.In previous in vitro studies, P. aeruginosa has shown the ability to display tolerance and resistance to antibiotics commonly used to treat PJIs. Although the Gram-positive Staphylococci are the most commonlyisolated pathogens from PJI, the infecting organism (or organisms) may notbe cultured as much as up to 25 % of the time or treatment isstarted before culture data are available. Thus, in treating PJI,combinations of antibiotics are commonly used to provide broad-spectrumcoverage . Vancomycinand/or aminoglycosides (most commonly gentamicin or tobramycin) can be mixedinto polymethyl methacrylate (PMMA) bone cement and mineral-based absorbable bone fillers such as calcium sulfate dihydrate (CaSOP. aeruginosa Xen41 (PA-Xen41) showed that the number and spacing of ALCS-B were important in the rate and extentof killing of an agar lawn biofilm. Further, we have previously shown in a similar model that theantibiotic loading concentration in ALCS-B did not significantly change theeffective area of antibiotic activity over the observed time frame, fromwhich we concluded that the area of antibiotic potency was limited primarilyby diffusion, not the loading potency. While we expect that increasing the area of spread of antibioticdepots will result in increased spatial coverage of antibiotic activity, wewish to determine the spatial effect of adding ALCS-B to an antibiotic-loaded spacer mimic on the spread of bacteria from biofilm-colonized materials, as well as the killing efficacy of biofilm bacteria on thosematerials in a scaled-up in vitro model. In the present study, we evaluate the effect of ALCS-B and antibiotic-loaded bone cement spacers (ALBC-S) on containing both the spread and killing of P. aeruginosa biofilms using a “large agar plate”model. We used P. aeruginosa Xen41 in part because of its strong bioluminescent signal, allowing for more sensitive and illustrative monitoring of the effect ofantibiotic diffusing from the PMMA or ALSC-B by an in vitro imaging system (IVIS).Previous studies using in vitro biofilms of the bioluminescent 22.1Pseudomonas aeruginosa PAO1 was used. Stock culture was streaked onto 1.5 % tryptic soy agar  and incubated for 24 h. A singlecolony was transferred aseptically to 15 mL of tryptic soy broth  and incubated overnight at37 P. aeruginosa isa relevant Gram-negative PJI pathogen.A bioluminescent derivative strain of 2.2Overnight cultures were diluted to 0.1 % and used to inoculate sterile,circular discs  of hydroxyapatite (HA),ultra-high molecular weight polyethylene (UHMWPE), 316L stainless steel(SS-316), and titanium (Ti). The “as received” roughnesses measured by contact profilometry were 976, 3867, 224, and 300 nm, respectively.The discs had a diameter of 9.5 mm and a thickness of 2 mm. Four milliliters of the diluted culture was added to four different wells of a six-well plate andthree discs of each material were aseptically submerged in the inoculum. Theplate was incubated at 37 2.3® Rapid Cure 10-cc mixing kits. Twenty grams ofCaSOALCS-B were prepared using Stimulan2.4PMMA bone cement spacer mimics were prepared usingSimplex™ P SpeedSet™Radiopaque Bone Cement construction kits , which are commonly used clinically. Antibiotic-loaded spacers were fabricated using 2 g VAN powder, 2 g TOB powder, and 40 g PMMA powder. Unloaded spacers were prepared with onlyPMMA bone cement powder. Twenty milliliters of sterile methyl methacrylate monomer (liquid) was added to the powder mixture, stirred into a dough-like mass, formed into a “hockey puck-like” disc using a 7 cmdiameter circular mold  and allowed to set for 30 min at 20 2.5Fifty milliliters of 1.5 % TSA was added to a 21 cm diameter glass pie baking dish sterilized with 70 % EtOH and allowed to solidify, followed by centralplacement of a PMMA spacer followed by another 100 mL of molten agar. Onceset, three discs of each material  were placed on top of the agar layer radiatingoutwards . For the experiments with ALCS-B, a 10-cc pack of beadswas sprinkled somewhat randomly around the spacer with greater bead densitycloser to the spacer mimic. While sprinkling the ALCS-B without carefulplacement in a specified pattern may cause difficulty with interpretation,we wished to more closely mimic how they might be applied clinically, wherethey might not be spread evenly. Also, small diameter antibiotic depots suchas ALCS-B can fill smaller void spaces than the larger PMMA beads and thuscan be distributed more thoroughly.After adding the beads, another 100 mL of cooled  that semi-quantitatively measures the relative amount of metabolically activebiofilm. Each quadrant of the large plate model was individually imaged andthen stitched together  to show the whole plate. Redrepresented the highest metabolic activity and blue or black low or nometabolic activity. White-light (plain) images of each plate were captured with a cellphone.2.7Colony-forming unit (CFU) counts were performed on mimicked sets of discs, one set on the 3 d biofilms and one set after incubation in the largeplate model. CFUs were performed as previously described.These discs were rinsed and then vortexed with 10 mL of phosphate-bufferedsaline  in 15 mLFalcon tubes ; 10 2.8All experiments were performed in triplicate. The effect of the differentconditions on the log reduction of biofilm at different proximities from thespacer was compared by first performing a log33.1P. aeruginosa Xen41 allowed biofilm on the discs and the spread ofbacteria from these discs to be easily visualized while remaining in situ in the large plate model over the 5 d . The unloaded spacer (US)condition of the large plate, containing no antibiotic, showed substantialbacterial spread from the discs and was confirmed by white-light imaging, showing P. aeruginosa coverage throughout the large plate .The bioluminescence of The antibiotic-loaded spacer (LS) alone exhibited anti-bacterial activity generating a zone of inhibition (ZOI) of approximately 2.4–3.0 cm from theedge of the spacer . After 3 d a small number of colonies were observed growing a few millimeters within the edge ZOI . These were possibly antibiotic tolerant phenotypes andhave been observed previously.The LS 3.2After 3 d of inoculation, the amount of PA-Xen41 biofilm grown onvarious discs was quantified by cell counting (CFU). This analysis wascompleted to confirm biofilm growth on each type of disc beforeimplementation into the large plate model. All four disc materials displayedsizeable biofilm growth of The LS alone reduced biofilm concentrations on the closest discs (1 cm fromthe spacer's edge), with bacterial reduction ranging from 1.5 to 5.5 log4P. aeruginosa Xen41 biofilm growth on materials common to orthopedicimplants in vitro while also increasing the zone of antimicrobial potency beyond that achieved by an antibiotic-loaded PMMA spacer alone. Furthermore, our work demonstrates how such beads sprinkled around the spacer may moreeffectively manage dead space and control the spread of bacteria from suchimplant surfaces, particularly in those locations where the spread ofreleased antibiotics is limited by diffusion, as would be expected in theperiprosthetic tissue, areas of a joint space, or the medullary canal. In awell-mixed system, the specific distribution of antibiotic depots will beless important since the antibiotics will be well distributed by the mixing,but when the rate of spread of antibiotics is limited by diffusion, then thespatial distribution becomes significant.Here we show in our diffusion-based model that ALCS-B significantly reduced. Antibiotics loaded into PMMA cement in the form of both beadsand spacers have been successfully used to treat PJI for many years. However, thereare limitations to the use of PMMA alone with respect to the release oflocal antibiotics at a surgical site. Once an antibiotic has eluted to belowMIC levels, the PMMA itself becomes a surface for bacterial biofilmformation. Further, even though retrieved PMMAspacers are still shown to elute antibiotics and produce a ZOI in laboratorytesting, the infection can persist, suggesting that the zone ofantimicrobial potency may be limited.Antibiotic-loaded PMMA beads have been used to increase the effective area of antibiotic activity, but they have limited penetration into small spacesand require surgical removal. Additionally, their relatively large size discsor fell farther away. This resulted in variability of killing efficacy. Ifbeads fell close to a biofilm-colonized disc, then there was a significant log reduction or eradication to below-detection limits, while discs furtheraway from beads exhibited less or no reduction. This spatial variation isevidenced by some of the large error bars  and illustrates theimportance of adequate spatial coverage within a periprosthetic joint orother infected surgical sites. Nevertheless, our in vitro data as well as those found clinically, and data from other in vitro studies  support theuse of ALCS-B as an effective means of releasing antibiotic to preventbiofilm spread and kill pre-grown bacterial biofilms.We recognize limitations in our study. Our study was conducted with only onebacterial strain. While we have shown similar efficacy with other Gram-negative and -positive species with respect to prevention and killingefficacy by antibiotics released from beads alone, to add rigor to our conclusions, we would need toinclude more pathogenic species and clinical strains. In addition, we used arelatively young biofilm grown for 3 d which may be less antibiotic-tolerant and less clinically relevant than more mature biofilms. Also, ourin vitro model is simplistic compared to the complex mechanical and chemical environment of surrounding bones, particularly that of an articulating humanjoint space. By design our model lacks any fluid flow, which would beexpected to enhance the spread of antibiotic from the spacer mimic and theALCS-B while also removing antibiotic from the system . Apharmacokinetic study by Hayes et al. (2014) found the intra-articular concentration of gentamicin releasedfrom a sponge in a dog dropped rapidly from approximately 2400to 4 The results from our in vitro study may support the adjuvant use of ALCS-B in the management of PJI, particularly for the release of antibiotics to theinterstices of the periprosthetic joint space in light of limited PMMAdiffusion with compromised vasculature. However, further work is required inorder to determine how our in vitro findings relate to the spread of antibiotics in the human joint or may relate to clinical outcomes. Such studies are nottrivial since measuring antibiotic concentrations at different locations intissues and fluids in a joint space requires multiple samplings at differenttimes, since sampling of joint fluid may not represent conditions everywherebacteria or biofilm may be present. We are currently developing a continuousflow reactor system which can accommodate clinically relevant antibiotic-loaded spacer mimics and beads, where we match the flow rate with reportedclinical drainage rates following TKA revision. Such a system may providefurther insight into the complex interplay between antibiotic releasekinetics from various administration methods, including addition ofantibiotic powder.