To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass. Bile is synthesized by the liver and stored in the gallbladder. In response to ingestion of a meal, the gallbladder contracts and empties bile into the small intestine resulting in the delivery of bile salts that aid in the assimilation of dietary fat. Despite an intense interest in the influence of intestinal microbiota in health and disease, it is surprising that there are few studies investigating the culture-independent microbiome of bile –11, noneE. coli and Enterococcus spp. in anywhere from 2.7 to 67%  of cases [Recently, a unique gallbladder disease in dogs has gained broad recognition –29. The of cases , 31–34. Studies examining the bile microbiome in humans are far from any consensus. Conclusions range from there being no bile microbiome  to the eOur objective in this study was to apply next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Our specific aims were to determine if a core bile microbiome could be identified in healthy dogs and whether dogs with mucocele formation were systematically colonized by previously unrecognized bacterial taxa. Integral to our study was inclusion in the analysis of (1) two independent DNA isolations on each sample, (2) two different extraction methods, and (3) sequencing of negative controls from different stages in the process, enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization performed on gallbladder mucosa.Apparently healthy purpose-bred research dogs were scheduled to undergo euthanasia for purposes of colony depopulation unrelated to this study at which time tissues became available for collection. Dogs relinquished to animal control and undergoing euthanasia for reasons related to aggressiveness toward humans or other dogs were sampled when available. Immediately after intravenous injection of a lethal dose of pentobarbital and auscultatory confirmation of cardiac arrest, the abdomen was opened via right flank abdominal incision without sterile preparation. With the incision held open, the gallbladder was visualized in the cranial abdomen and aspirated using a 12 cc syringe with 18-gauge needle without contacting the body wall or other internal organs. Bile was transferred within 10 minutes to a sterile microcentrifuge tube and frozen in liquid nitrogen prior to storage at -80°C. The gallbladder was subsequently removed from the abdominal cavity and a 1 x 1 inch square sheet of mucosa was excised and placed in 10% neutral-buffered formalin.Dogs with gallbladder mucocele formation were client-owned patients of either of two participating specialty referral hospitals where mucocele formation was diagnosed by means of ultrasonographic criteria  and owneThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of North Carolina State University. (Protocol Number: 14–049).Bile samples collected from control dogs underwent cytological examination by an American College of Veterinary Pathology certified study investigator (D.S.). Each cytological specimen was prepared at the time of bile collection by smearing a drop of bile onto each of 2 individual glass microscope slides. Slides were air-dried, fixed, and stained with hematoxylin and eosin. Samples of gallbladder mucus from dogs with mucocele formation were not examined cytologically.2 (blood agar and MacConkey agar) or in an anaerobic chamber (BBE/LKV and enrichment). All cultures were examined for growth daily for up to 5 days. Bacterial isolates were isolated and identified by laboratory standard operating procedures, including MALDI-TOF. At least one subculture of enrichment was performed during the growth period.For aerobic and anaerobic culture, samples of bile or mucus were directly plated on Columbia agar with 5% sheep blood, MacConkey agar  and pre-reduced Brucella blood agar plates . Additionally, samples were enriched in chopped meat broth. All plates and enrichments were incubated at 36°C in 5% COMedical records of client-owned dogs were reviewed for collection of age, breed, sex, neuter-status, and history of antibiotic administration within the prior 2 week period. Ultrasonographic images of the gallbladder were reviewed by an American College of Veterinary Radiology certified study investigator (G.S.) and histology findings from submitted surgical samples of the gallbladder were reviewed to confirm the final diagnosis of gallbladder mucocele formation.in situ hybridization (FISH) was performed as previously described [5’-GCTGCCTCCCGTAGGAGT-6-FAM-3’, where 6-FAM is 6-carboxyfluorescein) and a negative-control non-Eub probe (5’-Cy3-CGACGGAGGGCQTCCTCA-3’). The probes were reconstituted with sterile water and diluted with hybridization buffer to final working concentrations of 5 ng/μl. Formalin-fixed and paraffin-embedded intestinal tissue from a piglet diagnosed with necrotizing enterocolitis was included in each hybridization experiment as a positive control.Formalin-fixed samples of gallbladder mucosa from control dogs and dogs with mucocele formation were embedded in paraffin, sectioned at thickness of 4 μm, mounted on poly-L-lysine coated slides, and fluorescence escribed , 38. ProTo aid in distinguishing genuine signals from reagent-derived signals, we performed two separate DNA isolations on each sample using reagents from kits produced by different suppliers . All DNAOne tube for each sample underwent further extraction using a “no-phenol” (NP) method while the other tube was extracted using a “phenol” (P) method. No-phenol samples were centrifuged for 1 minute at 15,000×g and the supernatant transferred to ½ Inhibix tablet (Qiagen) and vortexed to fully disperse the tablet. The sample was centrifuged at 15,000×g for 2 minutes. The resulting supernatant (300 μl) was mixed well with 300 μl AL buffer (Qiagen) in a clean tube and incubated at 55°C for 8 hours. After incubation samples were mixed well with 300 μl of 100% ethanol and loaded onto a Zymo-Spin IC silica mini-elute column (Cat#C1004-250) and centrifuged at 8,000×g for 1 minute. Columns were washed with 500 μl of AW1 wash buffer (Qiagen) and centrifuged for 1 minute followed by 500 μl of AW2 wash buffer (Qiagen) and centrifugation for 5 minutes. Columns were dried for 10 minutes and DNA was eluted by incubation with 25 μl of PCR-grade water for 5 minutes followed by centrifugation for 2 minutes at 8,000×g.Duplicate samples extracted using the phenol method were treated with 500 μl of phenol-chloroform and vortexed for 5 minutes. Following centrifugation 10,000×g for 2 minutes the aqueous phase was transferred to a new tube to which 500 μl of chloroform was added followed by vortexing and centrifugation. The aqueous phase was similarly washed again in chloroform prior to transfer to a new tube. Washed samples were mixed well with 2× volume Zymobiomics binding buffer, applied to a Zymo silica column, and centrifuged for 1 minute. The column was washed twice with 500 μl of Zymo wash buffer and allowed to dry for 10 minutes. DNA was eluted from the column by incubation with 25 μl of PCR-grade water for 5 minutes followed by centrifugation for 2 minutes at 8,000×g.For each extraction method (no phenol versus phenol), 6 extraction control tubes treated with only PCR-grade water and lacking the addition of bile or mucus were included in all steps. For each extraction method, 3/6 tubes had a collection swab inserted to account for potential DNA contamination introduced by the swab.™ PicoGreen® dsDNA Reagent , and normalized prior to equimolar pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge MiSeq instrument (Illumina). Automated cluster generation and paired–end sequencing with dual reads were performed according to the manufacturer’s instructions.Extracted DNA was quantified via PicoGreen analysis and used for bacterial 16S rRNA gene amplicon sequencing as described , 40, witCanis lupus familiaris reference Cf31, the Canis lupus mitochondrial reference, or the Human Hg19 reference were eliminated. The resulting paired-end reads were processed with the QIIME 2 2018–6 [Sequencing output from the Illumina MiSeq platform were converted to fastq format and demultiplexed using Illumina Bcl2Fastq 2.20.0.422. Paired end reads were classified with Kraken2 [2 2018–6  wrapper 2 2018–6  includin2 2018–6 . Results2 2018–6  database2 2018–6  in QIIME2 2018–6  in QIIME2 2018–6  and Bray2 2018–6  between Taxa amplified from any negative control samples under any extraction conditions were designated as DNA contaminants. Contaminating taxa were excluded from patient sample analysis when present at a read count < 10 or ≤ the maximum read count observed in any negative control samples. As described by others working with sequence data from bile samples , taxa noFor the purpose of this study, members of a core microbiome were defined as ASV not designated as a DNA contaminant that were amplified using both extraction methods from the same sample, and detected in at least 30% of samples.Canis lupus familiaris reference Cf31, the Canis lupus mitochondrial reference, and Human Hg19 reference accounted for a median of 44 reads per control sample  and 709 reads per mucocele sample . These reads represented a median of 0.113% of total raw reads  in control samples and 2.3% of total raw reads  in mucocele samples. The resulting average and per sample raw and post-processing read counts and total ASVs identified under each extraction condition in experimental and negative control samples are shown in To enable identification and filtering of expected reagent and laboratory DNA contaminants from analysis of gallbladder bile and mucus sample sequencing results, negative extraction control samples were processed through two DNA isolation conditions and subject to 16S rRNA gene amplicon sequencing. Reads aligning against the A total of 108 different ASVs were amplified from ≥ 1 negative extraction control sample and therefore designated as laboratory or reagent DNA contaminants . SeventyBile samples were collected from 13 apparently healthy control dogs. Ten dogs underwent euthanasia by animal control and 3 were purpose-bred research dogs euthanized for reasons unrelated to the study. Represented breeds were Staffordshire terrier mix (7 dogs), Hound (3 dogs), Beagle, Border Collie mix, and German Shepherd (1 dog each). Median age was 2.5 years .After filtration of contaminating sequences and taxa with read counts ≤10 in all samples, control bile samples yielded a median of 3,686 reads  using the non-phenol extraction method and a median of 1,439 reads  using the phenol extraction method. A total of 94 unique ASVs were identified among all control bile samples. Sixty-seven (71%) of these ASVs were not amplified from any negative extraction control samples .The remaining 27 ASVs were also amplified from ≥ 1 negative extraction control samples and were retained in the analysis because their read count in bile exceeded the maximum count observed in any negative extraction controls .Stenotrophomonas . Three taxa were observed in 3/13 control bile samples and included Bacillus halodurans  and a member of the genus Novosphingobium  and genus Massilia .The majority of ASVs in Spirochaetaceae , genus Novosphingobium , genus Bacillus , Bacillus halodurans  and genus Veillonella .The number of unique ASVs per bile sample ranged from 2 to 9  using the non-phenol extraction method and 1 to 17  using the phenol extraction method . Only 5 Bacteria were not identified by cytological examination in any control dog bile samples. Yeast organisms were observed in 2 control bile samples (sample 7 and 19). Bacterial growth was not observed in any control dog bile samples under either aerobic or anaerobic culture conditions. Histological examination of the gallbladder was performed in all control dogs, all of which appeared unremarkable and none of which had eubacteria identified by FISH. Combining cytology, conventional culture, and FISH, the presence of bacteria was demonstrated in samples from 0/13 (0%) control dogs.Samples of gallbladder mucus were collected from 13 dogs with mucocele formation . RepreseAfter filtration of contaminating sequences and taxa with read counts ≤10 in all samples, mucus samples yielded a median of 5,915 reads  using the non-phenol extraction method and a median of 8,921 reads  using the phenol extraction method. A total of 109 unique ASVs were identified among mucocele mucus samples. Seventy-eight (71%) of these ASVs were not amplified from any negative extraction control samples .The remaining 31 ASVs were also amplified from ≥ 1 negative extraction control samples and were retained in the analysis because their read count in mucus exceeded the maximum count observed in any negative extraction controls .Lactobacillus brevis  and members of the genus Peptoniphilus  and Mitochondria . In no dogs was the presence of these 3 taxa confirmed in the sample using both extraction methods.The majority of ASVs in Escherichia coli/Shigella (samples 13 and 26).The number of unique ASVs per mucus sample ranged from 0 to 23  using the non-phenol extraction method and 0 to 19  using the phenol extraction method . Only 4 E. coli (3 dogs) and Enterococcus spp. (1 dog). Parallel results of 16S rRNA gene amplicon sequencing confirmed presence of the cultured bacterial isolate in 3/4 culture positive dogs and bacteria were observed by FISH in 2/3 culture positive dogs that had FISH performed  or Streptococcus  mucus samples and included erformed . For thepositive). The remprotocol  Tables.At a rarefaction depth of 1,000 reads, there were no significant differences in alpha diversity measures of evenness (Shannon), diversity (Faith), or observed number of ASV between samples of gallbladder bile from healthy dogs and samples of mucus from the gallbladder of dogs with mucocele formation. Both weighted and unweighted measures of beta diversity identified a significant difference in composition of ASV between control and mucocele gallbladder samples (Bray-Curtis PERMANOVA q = 0.002). Extraction method was observed to significantly affect only non-phylogenetic based differences in beta diversity (Bray-Curtis PERMANOVA q = 0.015).This study is the first to characterize the culture-independent microbiota composition of bile in healthy dogs using next-generation sequencing. Comparable studies in healthy people are lacking because of the invasive nature of bile sampling and are therefore limited to bile collected from patients with biliary disease –11, fromStenotrophomonas, Novosphingobium, Bacillus halodurans, and Massilia are worthy of brief discussion as they were observed in samples from several healthy control dogs. Each of these taxa were amplified at a low read count, were inconsistently validated by recovery using both extraction methods, and are well-recognized as either environmental extremophiles or water and soil-associated bacterial contaminants of sequencing studies [While contaminating ASVs were common in sequence data from bile samples, the majority of ASVs identified in bile from healthy dogs did not overlap with ASV observed in negative control extractions. Nonetheless, most of these ASVs were identified in only 1 or 2 bile samples. These findings do not support the existence of a core bile microbiota. Absence of a core microbiota is also supported by concurrent negative results of aerobic and anaerobic bacterial culture, cytological examination of bile, and eubacterial FISH performed on gallbladder mucosa. What remains unclear is whether these ASVs identified in bile from healthy dogs reflects genuinely endogenous bacterial DNA or an unaccounted for source of additional contamination. If genuine, our findings suggest that genesis of this microbiota would have to be unique to each individual and seemingly random. Notably, within each bile sample, amplification of individual taxa was not reproducible when comparing results of the 2 different DNA isolation methods. This lack of reproducibility is considered a hallmark of 16S rRNA sequence contamination . It is i studies , 52–55. Enterococcus spp. and E.coli-Shigella were common and abundant contaminants of the extraction materials and reagents. This presents a major limitation to the use of 16S sequencing approaches for distinguishing infection versus contamination with these bacteria or their DNA in bile samples. In some instances in this study, the presence of these bacteria could have been missed simply because low copy number of 16S rRNA gene sequences fell within the expectations of background contamination. A single control dog in the study had a large abundance of E. coli-Shigella and Streptococcus sp. sequences in bile consistent with likely infection. It is possible that this dog was a false-negative for bacterial culture or the sample had an unusually high level of contamination. Because the history of apparently healthy shelter dogs was unknown, occult bile infection of this dog is possible.Unexpectedly to us, 16S rRNA gene sequences for well-recognized biliary pathogens such as E. coli or Enterococcus spp. were obtained for 30% of gallbladder mucus samples. Escherichia coli and Enterococcus spp. are the most common bacterial causes of cholecystitis in dogs [E. coli, Clostridium perfringens, and Streptococcus spp.) from gallbladder mucus. In each positive instance, FISH demonstrated the bacteria as largely confined to superficial layers of the gallbladder mucus. Collectively, these findings support prior observations that gallbladder mucoceles can become infected by common enteric bacteria [The second aim of our study was to determine whether the gallbladder of dogs with mucocele formation is systematically colonized by any uncultivable bacterial taxa that could possibly play a role in disease pathogenesis. To date, evidence suggesting a role for bacteria in the primary pathogenesis of gallbladder mucocele formation in dogs has been largely circumstantial. Retrospective studies identify that a median of 13% of dogs with mucocele formation have positive bacterial culture results of gallbladder content , 31–34.  in dogs  and the  in dogs , 31–34.  in dogs . In thisbacteria , 31–34. Geobacillus, a recognized contaminant [Ochrobactrum [Sphingobium [Bacillus [Peptoniphilus and Lactobacillus brevis were each identified in 4 dogs. Peptoniphilus is recognized as a contaminant [Lactobacillus brevis. Amplification of Lactobacillus brevis was not restricted to one extraction method, however failure to simultaneously demonstrate this ASV in any mucus sample by both extraction methods is a trademark of DNA contamination. Collectively, these findings do not support the existence of a core mucus microbiome in the gallbladder of dogs with mucocele formation nor identify any taxa that are systematically associated with this disease.A range from 0 to 23 different uncultured ASVs were identified by 16S rRNA gene amplicon sequencing of mucus samples from dogs with gallbladder mucocele formation. Roughly 28% of these ASVs were also amplified from negative extraction controls and therefore consistent with documented reagent and laboratory DNA contaminants. High within-sample abundance was observed for some ASVs such as taminant , 58 whicobactrum , Sphingoingobium , 57, andBacillus , 53, 57 It is remarkable that 72% of the ASVs identified in the gallbladder mucus of dogs with mucocele formation were different in identity to ASVs observed in control bile samples. It is possible that this reflects predilection of the gallbladder for different consortia of bacteria in the setting of mucocele formation. But it is reasonable to consider that the difference could be attributed to differences in contaminants introduced during the sample collection process. Control bile samples were collected with a syringe and needle directly from the gallbladder, while mucus samples were collected with surgical instruments after removal of the gallbladder and incision through the gallbladder wall. Limitations of our study are lack of inclusion of technical controls that would have enabled confirmation of contaminants introduced during sample collection. Other limitations are potential confounding effects of antibiotic administration in some dogs with gallbladder mucocele formation which is an unavoidable circumstance when studying clinical patients. A history of antibiotic administration was not associated with any obvious discordance between culture, FISH, and 16S sequencing results for dogs in the study.In conclusion, this study does not support the existence of a core microbiome in the bile of dogs nor any systematic influence of uncultured bacteria on pathogenesis of gallbladder mucocele formation. Our elected approach of using 2 different DNA extraction methods for parallel 16S rRNA gene amplicon sequencing of each sample and sequencing of negative control extractions enabled identification of authenticated and putative contaminating DNA sequences that was essential to interpretation of these findings.S1 TableData represent the average number of reads and average % abundance of 3 replicates for each condition.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableResults of conventional cytology, aerobic and anaerobic bacterial culture, and eubacterial fluorescence in-situ hybridization (FISH) are also shown for each sample.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableResults of aerobic and anaerobic bacterial culture, and eubacterial fluorescence in-situ hybridization (FISH) are also shown for each sample.(DOCX)Click here for additional data file.


The potential heritability of DNA methylation prompted us to investigate the following question: Is the enhanced axonal regeneration phenotype inherited transgenerationally without exposure to folic acid supplementation in the intervening generations? In the present review, we condense our findings showing that a beneficial trait  and accompanying molecular alterations , triggered by an environmental exposure  to F0 animals only, are inherited transgenerationally and beyond the F3 generation.Human epidemiological studies reveal that dietary and environmental alterations influence the health of the offspring and that the effect is not limited to the F1 or F2 generations. Non-Mendelian transgenerational inheritance of traits in response to environmental stimuli has been confirmed in non-mammalian organisms including plants and worms and are shown to be epigenetically mediated. However, transgenerational inheritance beyond the F2 generation remains controversial in mammals. Our lab previously discovered that the treatment of rodents (rats and mice) with folic acid significantly enhances the regeneration of injured axons following spinal cord injury  Nearly 20 years ago, our neuroscience laboratory discovered that parenteral folic acid administered for 3 days before spinal cord injury enhances the regeneration of sensory spinal axons in a peripheral nerve placed at the site of a sharp spinal cord injury  1]. Sur. Sur1]. Whereas the developing mammalian CNS is able to generate and extend neurons , the difTo test the contribution of glial vs. neuronal factors to folic acid–mediated axonal regeneration after injury, the left lumbar DRG of rats with injury to the left sciatic nerve and both dorsal columns were harvested after folic acid pretreatment, dissociated, and placed in a culture medium permissive of axonal growth to establish a pure neuronal culture devoid of glia  We obserFolr1 (Folr1+/−) reduces the spinal axonal regeneration capacity compared to wild-type controls in response to folic acid supplementation [Transport of folates across epithelial membranes and into cells is mediated by three transporters : the folentation . Proton-entation . Next, wentation . These dde novo DNA methylation. To test whether the DNMTs modulate folic acid–mediated axonal regeneration, we used immunoblot assays to measure the protein expression with escalating folic acid doses. We observed no difference in DNMT1 levels following spinal axon injury and folic acid supplementation. However, the protein levels of DNM3A and DNMT3B were significantly suppressed by injury and restored to baseline with folic acid supplementation. Moreover, with increasing doses of folic acid, DNMT3A/B expression followed a biphasic curve in parallel with that observed in spinal axon regeneration  folate congener to control for specificity of methyl donor agonist. This folate agonist does not require DHFR conversion to be activated. Also, a non-parenteral diet supplemented with folic acid, betaine, choline, and vitamin B12 (Harlan Laboratories Diet 7017-MS) was also provided to mating F0 breeding pairs. Both the parenteral L-methylfolate and the oral methyl-supplemented diet independently enhanced spinal axon regeneration to at least four successive untreated generations (F1–F4). These results indicate that the transgenerational axonal regeneration phenotype is specific to methyl donor agonism but not to its specific analog or route of administration.in vivo and in vitro experiments [Next, inbred Fischer rats were used to test whether the transgenerational post-injury–enhanced axonal regeneration phenotype is specific to a rat strain . We obseeriments . Most teeriments . To our eriments , furtherin vivo vs. in vitro experimental models, outbred or inbred rodent strain, or rodent genus.Together, these data indicate that the TEI of the enhanced axonal regeneration phenotype is not an artifact of folate analog, route of administration, P-value < 1e-5) and 782 differences in mRNA transcript levels in spinal cord tissues between lineages with and without ancestral F0 folic acid. Transgenerational changes in both DNA methylation and mRNA expression levels were observed in 13 genes [Ank3, Cntf)  and histone deacetylase (trichostatin A) to the F3 generation and beyond. Both blocked the pro-regenerative effect of ancestral folate [13 genes . Methyla13 genes . Using s3, Cntf) . A compade novo DNMTs. The folate-enhanced axon regeneration phenotype is surprisingly heritable via non-Mendelian patterns of inheritance in tandem with differential regions of DNA methylation, hydroxymethylation, and differential RNA expression.Single-carbon pathways support the capacity of injured spinal cord axons to regenerate after injury. In turn, the beneficial effects of folate administration require a fully functional methylation cycle. The effects of folate on regeneration of spinal afferent neurons are biphasic and dose-dependent in correlation over a wide dose range with global and gene-specific DNA methylation and with the activity of 9) is an essential nutrient required for a diversity of enzymatic reactions and processes in a broad spectrum of biological pathways, including DNA replication, nucleotide and amino acid synthesis, metabolism, and methylation reactions [Folate , different genera, and other injury models that may comprise benefits to progeny after ancestral single-carbon supplementation.The molecular mechanisms underlying non-genomic inheritance are incompletely resolved, and the concept of mammalian TEI remains open to controversy . AccordiCreb1, Dscam, Emb, and Runx3 [Although we have only just begun to glimpse the mechanisms that underlie the beneficial effects of folates on axon regeneration after injury, we note that neuronal trauma appears to dysregulate folate pathway receptors and enzymes while simultaneously increasing the requirement for enzyme cofactors and methylation of gene promoters. A subset of genes with increased promoter methylation participate in conventional neuronal function pathways, whereas others such as nd Runx3  participnd Runx3 . Surprisnd Runx3 , 23–25.


Sedum CAM-species on mm-thin limestone pavements to the species-rich assembly of C-3 terrestrial plants in deeper and wetter soils. A GLM-model indicated that 90% of the variation in species richness could be explained by a positive effect of soil depth, a negative effect of the duration of water cover and their interaction. In water, mean photosynthesis was highest among aquatic species, low among Sedum species and cushion mosses, and negligible among C-3 terrestrial plants. While aquatic species dried out in air, drought-resistant small species were probably competitively excluded from the more suitable terrestrial habitats on deeper soils with moderate flooding by taller species of high photosynthetic capability. In conclusion, the clear distribution of species along the steep hydrological gradient reflects distinct structural and physiological adaptations, environmental filtering and interspecific competition.Plant species often separate strongly along steep environmental gradients. Our objective was to study how coupling between plant physiology and environmental conditions shapes vegetation characteristics along a distinct hydrological gradient. We therefore investigated species photosynthesis in air and under water within a limited area from dry-as-dust to complete submergence in a nutrient-poor limestone habitat on Öland’s Alvar, Sweden. We found structural and physiological adaptations of species to endure water limitation at the dry end  and diffusive carbon limitation  at the submerged end of the gradient. As anticipated, mean photosynthesis in air increased 18-fold from the species-poor assembly of cushion-mosses and  The fouTo evaluate how physiological and morphological traits change along the relative finely graded division of plant species into moisture preference groups  we used Spearman rank correlation due to the inherent character of the Ellenberg values . Correlations between continuous traits were correlated using Pearson product moment correlation after evaluation of the assumption of normality.Differences between plant groups with different overall photosynthetic and drought tolerance strategy  in photosynthetic rates were tested by means of an ANOVA test followed by Tukey’s post hoc test. Deviance from homogeneity of variance was assessed by Brown-Forsythe’s test. We did not test the morphological trait differences between these groups, as many representatives of the aquatic C3 and CAM + mosses cannot be meaningfully evaluated for root porosity and SLA.To evaluate how species richness was regulated along the investigated hydrological gradient we applied Poisson generalized linear models (GLM) with a log link function. The log link function ensures positive fitted values, and the Poisson distribution is commonly used for counts (such as species richness). The explanatory variables used were number of days with water cover and average sediment depth. Because the effect of sediment depth potentially depended on the water cover, we included an interaction term between the two variables in the model. The significance of the interaction term was evaluated by comparing nested models using analysis of deviance . Check fThroughout, variables not normally distributed, according to the Shapiro–Wilk test, were log-transformed to ensure normality. Alpha levels below 0.05 were considered significant. Correlations, ANOVA tests and all graphs were performed in GraphPad Prism version 8.4.3  while the Poisson GLM were conducted using R version 4.0.2  using th−1 DW−1 h−1 and rates were systematically low among the four drought-resistant cushion mosses (range: 15–45 µmol C g−1 DW− h−1) and the two succulent, constitutive CAM-species, Sedum album and S. acre (−4–39 µmol C g−1 DW− h−1) of low Ellenberg-F indices (2) on thin soils as adaptations to arid conditions (−1 DW− h−1) among thirteen terrestrial angiosperm species, including emergent plants in the shallow ponds, growing on thicker soils with better water supply (Ellenberg F indices: 3–10). Photosynthesis of terrestrial species in air increased highly significantly with their Ellenberg F index from 2 to 10  and higher specific leaf area . Submerged species (Ellenberg F of 11–12) rapidly desiccated when exposed to air [As hypothesized, photosynthesis in air showed a marked peak for terrestrial species of intermediate Ellenberg F-values located in the middle of the hydrological gradient, while values were very low for species at the dry end and zero because of leaf desiccation of aquatic species at the submerged end of the gradient A. Mean pnditions . Mean phSedum species . Additionally, higher Ellenberg F-values were significantly correlated to increasing specific leaf areas .The other key traits recorded at the species level also showed correlation with the Ellenberg F-values. Root porosity and Ellenberg F-values increased in concert and formed a highly significant positive relationship . The five terrestrial species did not raise pH and reduce CO2 much relative to air equilibrium and only extracted a minimum amount of the initial DIC pool . This result indicates that while species richness increased with sediment depth this effect was dependent on the duration of flooding at the given site. This interaction led to the highest species richness being found on pond banks with deep soil and limited flooding. The explained deviance of the model, or pseudo R2 [The large group of terrestrial C-3 species was widely distributed across the entire hydrological gradient, but most species was found at higher soil depth and low to intermediate water cover. Statistical analysis of species richness along the investigated hydrological gradient by means of a Poisson GLM regression showed a significant interaction between duration of water cover and sediment depth . At the wet end of the hydrological gradient, the group of aquatic plants  only occurred under permanently inundated conditions.The small CAM-species The environmental conditions at the study site ranged from extremely dry with plants growing in soils few mm deep above the solid limestone plates and experiencing summer temperatures above 40 °C, through more productive areas with deeper soil, higher availability of soil nutrients and water in limestone crevices or close to ponds, to permanent submergence within the ponds. Most findings concur with expectations, but we have expanded the analysis to a complete hydrological gradient and have evaluated the distribution of species in relation to their photosynthetic capability both in air and under water.Sedum species located at the arid end (Ellenberg-F: 2). We did not measure the growth rate of the twenty-three species, but may use photosynthetic rates as a proxy for this property [Sedum species have adapted to limited water availability by having small, densely packed succulent leaves and inducing CAM-metabolism under drought [Measured photosynthesis in air peaked at the mid to wet end of the hydrological gradient (Ellenberg-F: 8–10) among thirteen terrestrial C-3 species and, on average, dropped eighteen-fold to the four small cushion mosses and two property . It is lproperty . The veg drought . Their wSedum album can be explained by a combination of high tolerance to limited water supply on thin soils and competitive exclusion by taller and faster growing species at higher nutrient and water supply in deeper soils. Field observations showed denser stands and greener shoots of Sedum album at sites with intermittent surface flow of water across the limestone surface and extra nutrients from horse droppings. These results support the notion that Sedum album and other drought-resistant small species are excluded from areas with deeper soils by competitive exclusion from taller species. The distribution pattern of Sedum species resembles that of other stress selected species of low competitive ability  such asal flats  and smalal flats .Mosses on the solid limestone surface form dense cushions reducing wind exposure and evaporation . Moss cuUnder submerged conditions, species face two potential physiological problems. Both of them are caused by limited availability and diffusion rates of gases in water to support respiration and photosynthesis. Oxygen supply to respiration can be critical under water because oxygen concentrations are about 20-fold lower at air equilibrium and diffusion coefficients are 10,000-fold lower compared to air. Thin tissue, high surface permeability to gases, high tissue porosity and reduced respiration rates are the common anatomical and physiological adaptations to improve oxygen supply in response to submergence ,23,24. A2 to underwater photosynthesis is similarly restricted by the lower diffusivity and, in addition, by CO2 concentrations reduced below air equilibrium, while concentrations required to saturate photosynthesis of submerged species based on CO2-use alone are some ten-fold above air equilibrium [2 concentrations  are required to saturate underwater photosynthesis of amphibious and terrestrial species [Chara vegetation, other submerged species need to use bicarbonate because photosynthesis in the examined ponds elevated pH and depleted CO2 between 7 and 360 times below air equilibrium at noon on summer days. These CO2 concentrations are close to or markedly below the CO2 compensation point of submerged species confined to CO2 use alone, limiting their photosynthesis and preventing growth [2 by coupling bicarbonate use (HCO3−) to precipitating calcium carbonate (CaCO3) on their outer surfaces allowing continued photosynthesis without further rise of pH in the water  + CaCO3 (precipitated); Sand-Jensen et al., 2021). Terrestrial species are excluded from the ponds, unless they can reach above the water surface, because they only extracted 0.6–1.3% of the DIC pool in the water and have CO2 compensation points above air equilibrium. The terrestrial species are unable to use bicarbonate and precipitate calcium carbonate .Supply of COilibrium ,16. Even species . Thus, ig growth ,19. The Chara-species collected from alkaline ponds varying from oligotrophy to eutrophy reported mean photosynthetic rates (110 µmol O2 g−1 DW h−1) that were three-fold lower than mean rates of the terrestrial C-3 species from the oligotrophic limestone site [The ability of many submerged species to use bicarbonate and thrive under water comes with an investment cost to produce the necessary transport proteins and catalytic enzymes and running costs to operate the ion transport . Likely one site .Sedum spp.) and diffusive carbon limitation at the submerged end  of the hydrological gradient in the limestone habitat. The species-rich assembly of terrestrial C-3 species was confined to better water and nutrient supply for roots in deeper and wetter soils as well as air contact for leaves in the mid to wet part of the hydrological gradient. These terrestrial C-3 species desiccated under drought and were unable to photosynthesize under water. Under optimal conditions, they benefitted from eighteen-fold higher mean photosynthesis compared to drought-resistant species and six-fold higher photosynthesis compared to submerged species. Drought-resistant species were likely competitively excluded from the more suitable habitats by taller and faster growing species. The distinct species distribution can be explained by the steep hydrological gradient, the specific structural-physiological adaptations combined with strong environmental filtering and interspecific competition. These relationships and interactions became highly distinct by examining species distribution and structural-physiological traits along a full hydrological gradient from dry-as-dust to full submergence.In conclusion, we found highly distinct structural and physiological adaptations to endure water limitation at the dry end (e.g., CAM-metabolism of


Programmed cell death has long been characterised as a key player in the development of human disease. Necroptosis is a lytic form of programmed cell death that is universally mediated by the effector protein mixed lineage kinase domain-like (MLKL), a pseudokinase. MLKL's activating kinase, receptor interacting protein kinase 3 (RIPK3), is itself activated within context specific scaffolds of receptor interacting protein kinase 1 (RIPK1), Z-DNA Binding Protein-1 (ZBP1) or TIR domain-containing adaptor inducing interferon-β (TRIF). These core necroptosis modulating proteins have been comprehensively revealed as potent drivers and suppressors of disease in inbred mouse strains. However, their roles in human disease within the ‘real world’ of diverse genetic backgrounds, natural infection and environmental challenges remains less well understood. Over 20 unique disease-associated human germline gene variants in this core necroptotic machinery have been reported in the literature and human clinico-genetics databases like ClinVar to date. In this review, we provide an overview of these human gene variants, with an emphasis on those encoding MLKL. These experiments of nature have the potential to not only enrich our understanding of the basic biology of necroptosis, but offer important population level insights into which clinical indications stand to benefit most from necroptosis-targeted drugs. Apoptosis can be initiated by a range of intrinsic and extrinsic signals, and is commonly regarded as a ‘clean death’ characterised by the caspase-mediated disassembly of cells into highly phagocytosable, membrane enclosed bundles [independent form of programmed cell death that is characterised by the release of highly inflammatory cytokines, intracellular proteins, and nucleic acids into the extracellular space [Programmed cell death takes many forms and is crucial to every aspect of normal animal development and homeostasis. The most well studied programmed cell death pathway is the caspase- bundles ,2. Unlikar space . In turnar space .Importantly, several additional, highly context-dependent facets to MLKL function have been reported . These Mlkl gene knock-out and knock-in mutant mice have revealed necroptosis as a driver or protective factor in murine disease spanning every bodily system [MLKL have revealed neurological and metabolic pathologies where the absence of MLKL function plays a driving role [de novo patient mutations in MLKL signal modulators like CASP8 and RIPK1 [MLKL [excessive necroptotic signalling is an important contributor to inflammatory disease  in two brothers diagnosed with progressive neurodegenerative spectrum disorder [. These data support gnomAD's ‘loss of function’ classification of p.Asp369GlufsTer22, making this the first peer-reviewed, clinical description of humans that are homozygous for an MLKL LOF gene variant. The brothers presented with an asymmetric lower limb weakness at the ages of 19 and 30, respectively, which developed into a novel progressive neurodegenerative disorder characterised by paresis, ataxia and dysarthria. Magnetic resonance imaging performed at 28 and 31 years, respectively, after onset of symptoms revealed severe global cerebral, cerebellar, cortical cerebellar fibers tract and brainstem atrophy and small, discrete periventricular T2-hyperintense white matter lesions. All pathology was limited to the nervous system and the patients did not exhibit any increased susceptibility to infections. It is important to note that this MLKL mutation also segregated with a homozygous in-frame deletion of one amino acid in the adjacent FA2H gene (fatty acid 2-hydroxylase), and a maternally inherited missense amino acid substitution in the X-linked gene AP1S2 (vesicle sorting and transport) in these patients. While FA2H variant function was not impaired when examined in vitro and the AP1S2 variant is classified as ‘benign’ based on ACMG guidelines [To date, only four individuals that are homozygous for a predicted loss of function (LOF) ancestry . In 2020disorder ,28. MLKLidelines , the infMLKL LOF gene variant  was found to be >20 fold enriched in a cohort of Hong Kong Chinese patients suffering from the neurodegenerative disease Alzheimer's, relative to a large ancestry-matched population [MLKL LOF variant is specific to populations of Southern Chinese descent, was only present in heterozygotes, and was not linked to any predicted damaging mutations in the adjacent FA2H gene. Wang et al. reported that shRNA-mediated knock down of MLKL in APPswe-293 cells led to an increased ratio of amyloid beta proteins Aβ42 to Aβ40. Further study of the precise role of MLKL in modulating levels of these proteins in neuronal cells remains an important area of future investigation.Another pulation . This MLMlkl gene knock-out (KO/KOMlkl) mice do not appear to exhibit the spontaneous, progressive neurodegeneration observed in the patients described by Faergman et al. [KO/KOMlkl mice also exhibit a reduced propensity for demyelination in several other independent experimental mouse disease models. This manifests as improved outcomes in mouse models of Parkinson's disease [MLKL LOF alleles is 0.0007,   . The ideMLKL missense variant p.G316D (rs375490660) was reported to be associated with Maturity Onset Diabetes of the Young (MODY) in a Palestinian family [ERN2 (endoplasmic reticulum stress), NIPAL4 (Mg2+ transporter activity) and SPTBN4 (actin binding). When compared with exogenously produced MLKLWT, protein levels of exogenously produced MLKL p.G316D are significantly reduced. RIPK3-phosphorylated (Ser358) MLKL p.G316D are basically undetectable despite normal upstream signalling. Exogenous expression of MLKL p.G316D was also unable to reconstitute necroptotic cell death in two lineage-distinct CRISPR-Cas9 KOMLKL/KO human cell lines and did not appear to influence the function of endogenously expressed MLKLWT in MLKL wild-type cells (simulating the heterozygous state of patients). While the stability and function of endogenously expressed MLKL p.G316D were not verified in patient-derived cells in its endogenous heterozygous context, normal levels of MLKL p.G316D mRNA in patient derived cells indicates that this missense mutation does not lead to reduced mRNA stability [MLKL p.G316D are yet to be investigated.Recently, a heterozygous n family . This vatability . For thi−5, MLKL p.G316D homozygotes have not been recorded in the gnomAD database. How a monoallelic reduction in necroptotic cell death contributes to the aetiology of diabetes in this family remains a matter of investigation. KO/KOMlkl mice do not display any of the hallmarks of diabetes when fed a standard laboratory mouse chow diet, and actually demonstrate improved glucose and insulin tolerance relative to Wt/WtMlkl controls when placed on a high fat diet for more than 12 weeks [Mlkl gene knockout or mutation is a genetic modifier in mouse models of spontaneous diabetes  could pMLKL LOF and missense variants to the incidence of MODY. With no ‘gold standard’ criterion or threshold for diagnosing MODY in human patients, broadening gene panels for precision genetic testing will inform clinical diagnosis and management [Importantly, such studies can also prompt examination of potential risk posed by other human nagement .MLKL gain of function or ‘activating’ mutations have been described both in vitro and in vivo in murine models [. Human MLKL however is less amenable to rationally designed modifications that confer RIPK3 independent activity [Mlkl was shown to result in systemic lethal neonatal inflammation in homozygotes and hematopoietic dysfunction in heterozygotes [Mlkl p.D139V, confers constitutive cell death activity to MLKL, removing the requirement of upstream necroptotic signalling and RIPK3-mediated phosphorylation. The human equivalent, MLKL p.D140V, has been observed only once, and only in heterozygous form in humans in the gnomAD database (rs1330913532). Interestingly, three very closely situated MLKL missense gene variants are amongst the most prevalent MLKL single nucleotide polymorphisms (SNPs) present in humans. These SNPs, MLKL p.Ser132Pro , p.Arg146Gln  and p.Gly*202Val - have not been previously associated with human disease in any previous genome-wide- or phenome-wide-association studies (GWAS or PheWAS) individually. They were however shown to occur in trans at 10–12 times the expected frequency in a cohort of patients suffering from chronic recurrent multifocal osteomyelitis (CRMO) [The biological consequences of spontaneous and rationally designed e models ,42,43. Hactivity . In 2020ozygotes . This mus (CRMO) . While tMLKL SNPs relative to wild-type MLKL is yet to be reported in the literature. However, the hypermorphic nature of the closely situated mouse mutation Mlkl p.D139V does raise important questions as to the evolutionary significance of such high allele frequencies. An estimated 8% of the global population are heterozygous carriers of MLKL p.S132P, p.R146Q or p.G*202V based on gnomAD data, and 167 are homozygotes. Many reported hypermorphic variants in immune-related genes that confer a survival advantage to specific pathogens have achieved high allele frequency in certain populations [An analysis of the cellular function of the protein products of these common human ulations ,46. ManyMLKL's implication in diseases like diabetes and Alzheimer's also warrants the investigation of any historical survival advantage conferred by MLKL gene variants or expression Quantitative Trait Loci (eQTL) with regards to nutrient acquisition and storage. An intriguing example of this was described recently for MLKL's upstream activator RIPK1. Human gene variants that increase RIPK1 gene expression were found to be associated with obesity [By this same evolutionary logic,  obesity . Nutriti obesity .MLKL gene variants have only been weakly associated with human traits or changes in disease risk in conventional GWAS. The highest 'P' value reported for an MLKL variant in a published GWAS was for a non-coding SNP rs2057805  located in an upstream regulatory region in adolescent idiopathic scoliosis [P-value of 3 × 10−9. Other notable associations of MLKL SNPs with quantitative human traits that are significant or approaching genome wide significance include one with comparative height at age 10 (P = 5.26 × 10−7) [P = 1 × 10−8 ) [P values <4 × 10−23 [MLKL and RIPK3 SNPs described indicate that they account for only very low proportions of the phenotypic variance in these quantitative traits, they nonetheless highlight the potential of such unbiased approaches for uncovering unexpected gene-function links [To date, coliosis . This as × 10−7)  and anotr 2021)) . Interes × 10−23 . While ton links .MLKL itself, but also variation in genes that can inappropriately unleash, amplify, or even dampen the cellular activity of MLKL. Based on observations of important MLKL regulatory genes gleaned from genetically modified mice [RIPK1, TNFAIP3 (encoding A20) and CASP8, where several unique LOF or missense mutations are characterised by autoinflammatory and lymphoproliferative syndromes [RIPK1, TNFAIP3 and CASP8 mutations in ClinVar does not equate to a larger contribution to the heritable variation in disease risk, when considering all disease burden in the human population. In fact, it is likely quite the opposite. Examining the mutational spectrum of ∼141 000 individuals catalogued by the gnomAD database permits a simple visualisation of the high evolutionary constraint on LOF variation in RIPK1, TNFAIP3 and CASP8 [MLKL and ZBP1 each exhibit a higher summed frequency of predicted LOF alleles than RIPK1, TNFAIP3 and CASP8 combined, and both are dotted with a larger repertoire of more common (>MAF 0.001) missense gene variants  that would cause them to be subject to purifying selection over time [MLKL, RIPK3 and ZBP1 genes also increases the statistical likelihood that these genes can act as low effect size modifiers of polygenic traits in humans  [When considering the pathophysiological impacts of necroptosis, we must not only consider genetic variation in ied mice , we haveied mice , as acceyndromes . It is ind CASP8   .CASP8 variant (p.R248W) in two siblings that presented with lymphadenopathy and splenomegaly. Caspase-8 p.R248W exhibited reduced stability and resulted in an enzymatically inactive protein, with peripheral blood lymphocytes defective in CD95-induced apoptosis. Caspase-8 activity is an important determinant of programmed cell death downstream of death receptor and inflammasome activation [L activity facilitates the stabilisation of RIPK1 and RIPK3 [L heterodimers). Amongst other signalling events, this also promotes the formation of the necrosome and MLKL-mediated cell death. Mice deficient in Caspase 8 die at embryonic day 10.5 but WT/KCasp8O heterozygotes are phenotypically normal [KO/KCasp8O embryonic lethality does not occur on a KO/KOMlkl background, but KO/KCasp8O, KO/KOMlklmice do go on to develop progressive lymphadenopathy [CASP8 variant -borne disease that do not manifest as ALPS.A full decade before MLKL's role in cell death was discovered, inherited deficiency of Caspase 8 was reported to mediate autoimmune lymphoproliferative syndrome (ALPS). Chun et al.  reportedtivation . The absnd RIPK3  ) that presented with diarrheal, perianal disease, failure to thrive and discontinuous severe proctocolitis, as well as increased susceptibility to bacterial and viral infections. Primary patient cells and cells engineered to express CASP8 p.Q237R exogenously exhibited increased IL-1β release in response to lipopolysaccharide (LPS) priming, that could be blocked by NLRP3-mediated inflammasome inhibitor MCC950 and the MLKL inhibitor necrosulfonamide. Furthermore, CASP8 p.Q237R, but not p.R265W, was also shown to predispose engineered HT-29 colon carcinoma cells to increased MLKL oligomerisation and death when exposed to a necroptotic stimulus [MLKL in this disease scenario is further supported by recent observations that MLKL fully mediates the ileitis and colitis observed in mice with intestinal epithelial cell-specific deficiency of Caspase-8 [CASP8 p.R248W/R265W also highlights that unchecked necroptosis may be variably expressed as a phenotype of the same CASP8 mutation, depending on the individual . The genetic or environmental determinants of this disparate role of MLKL remains an important line of future investigation into the potential toxicity of Caspase-8 targeted drugs.Caspase-8 deficiency manifests as very early-onset Inflammatory Bowel Disease (VEO-IBD) in some individuals. Lehle et al.  reportedaspase-8 . Chun etaspase-8  clinicalall of, the downstream effects born of genetically encoded RIPK1 dysfunction. In 2018, Cuchet-Lourenco et al. [RIPK1 LOF nucleotide deletions, and a number of additional patients with homozygous RIPK1 LOF alleles were reported soon after [Casp8, Ripk1 knockout in murine models is homozygous lethal, with mice dying during the immediate post-natal period due to systemic inflammation and cell death in multiple tissues. This inflammation is ameliorated in a KO/KOMlkl background, but mouse pup survival is only extended by a few days [ KO/KORIPK1 mice is only bypassed when both necroptosis and extrinsic apoptosis are removed from the equation by compound genetic crosses [RIPK1 LOF in humans can lead to severe disease, but is not incompatible with life as it is in mice. Is this difference intrinsic to inter-species differences in RIPK1 itself, or can it be explained by key species-specific differences in the regulation of downstream signal effectors like MLKL?Given the important position of RIPK1 at the nexus of several cellular signalling pathways, MLKL and necroptosis account for a significant portion, but not o et al.  reportedon after . Patienton after . An incron after ,71. Pation after . Similarfew days . The sev crosses . HomozygRIPK1 gene variants that prevent RIPK1 cleavage by caspases. Heterozygosity of such ‘cleavage resistant’ alleles is sufficient to induce a severe periodic inflammatory syndrome in humans characterised by fever and lymphadenopathy [KO/KORipk1 mice and for reasons that cannot be remedied by the deletion of Mlkl [Mlkl .Compounding these questions are disease causing missense enopathy . Homozyg of Mlkl ,78. The RIPK3 LOF gene variants. One individual that is homozygous for a predicted LOF RIPK3 allele was recorded as part of the ‘human knockout project’, though details of this individual's phenotype were not provided [RIPK3 p.Pro493ThrfsTer9 (rs531266348), contains a frame shift insertion that may lead to a short C terminal truncation as opposed to a full LOF. As such, its classification as ‘Loss of Function’ is classified as low confidence in the gnomAD database, as is the only other potential LOF variant found in homozygous form; RIPK3 p.Arg422Ter (rs146886719). Similar to the KO/KOMlkl mouse, KO/KORipk3 mice do not show any overt signs of spontaneous neurodegenerative disease or diabetes under steady state conditions, even when aged beyond 12 months [ KO/KORipk3 mice do show evidence of disturbed bone architecture and increased osteoclasts, supporting the reported association of the RIPK3 cis-eQTL rs3212240 with lower estimated bone mineral density in a human cohort [KO/KOMlkl and KO/KORipk3 mice show a significantly reduced propensity for reproductive organ aging [To date, there have been no clinical descriptions of humans that are homozygous for provided . The all2 months ,81. Inten cohort . Given oan aging . NecroptMLKL's role in heritable disease. Two distinct, de novo MLKL LOF variants have been shown to segregate with a severe familial neurodegenerative disorder and familial maturity onset diabetes of the young (MODY). While MLKL LOF alleles are more prevalent in the population relative to other key genes involved in programmed cell death, they are dwarfed in frequency (>100-fold less frequent) by a series of common non-conservative protein modifying mutations that cluster at key functional domains of MLKL. The identification and clinical description of additional families and individuals carrying homozygous and heterozygous MLKL LOF gene variants is not unlikely given the growing uptake of whole genome sequencing for clinical diagnosis. While rare, these individuals will reveal new and important insights into the role of MLKL and disease aetiology and the basic molecular mechanism of MLKL's varied cellular functions. They will help to focus and fine-tune the study of the trait- and disease- modifying potential of the MLKL protein coding variants carried by >10% of the global population, unlocking new clinical indications and contraindications for drugs that target necroptosis and its important upstream regulators.This review gives an overview of the current landscape of Naturally occurring human gene variants are important tools for the study of necroptosis at the molecular and pathophysiological level.de novo necroptosis gene variants have been implicated in autoinflammatory disease, neurodegeneration and metabolic disease.Rare and MLKL. Studying their role in human health and disease will uncover new clinical indications and contraindications for drugs that target necroptosis and its important upstream regulators.More than 10% of individuals carry protein-coding single nucleotide polymorphisms in


Tubby mice exhibit hearing impairment due to the loss of stereocilin from the tip regions that connect the tallest stereocilia of the outer hair cells (OHCs) to the tectorial membrane. Stereocilin is an essential stereociliary protein in the OHCs, the mutation of which in humans causes autosomal recessive non-syndromic deafness. Map1a is a modifier of tubby hearing (moth1), and its wild-type allele, rather than the moth1 allele from the C57BL/6 J strain, restores stereocilin localization to the stereocilia and rescues the hearing impairment of tubby mice. The mechanism by which MAP1A accomplishes this is unclear, partly due to ambiguity regarding whether the tubby mutation is a true null. We therefore generated Tub-null (Tub−/−) mice by deleting exon 3 and found that they exhibit hearing impairment like that of tubby mice, suggesting the tubby mutation is a loss-of-function mutation with regard to hearing. When we crossed Tub−/− mice with AKR mice that have wild-type Map1a alleles, we found that wild-type MAP1A restores stereocilin localization to the tips of stereocilia and rescues hearing impairment. These data suggest MAP1A does not require interaction with tubby protein in maintaining stereocilin at the tips of stereocilia and that OHCs use two independent molecules—MAP1A and tubby—to doubly ensure proper stereocilin localization.The online version contains supplementary material available at 10.1186/s13041-022-00966-z. Tubby mice show obesity, blindness and deafness [tubby mutation is a G-to-T transversion that causes a splicing defect in the 3’-end of the Tub gene [tubby mice have been remained mysterious for a long time. We recently revealed the molecular mechanism by which tubby mice develop hearing impairment. Stereocilin, which should be localized to the tips of auditory stereocilia, is mislocalized in tubby mice [Strc knockout (KO) mice have defective TMALs, which are essential for mechanotransduction, leading to hearing impairment [tubby mice phenocopy Strc KO mice [Map1a is a modifier of tubby hearing (moth1) [moth1 allele from C57BL/6 J mice, rescues the hearing impairment of tubby mice [deafness . The tubTub gene , but thepairment . Natural (moth1)  and, inttubby mice [Tub transcript and tubby protein were reported in the retina and the cochlear of tubby mice [Tub conditional KO mice in which exon 3 of the Tub gene is flanked by loxP sites. The resulting Tubflox/flox mice were crossed with a global “Cre-deleter” mice (E2a-Cre) to produce homozygous null offspring  mice also show hearing impairment. Immunofluorescence staining in Tub−/− mice we generated revealed that stereocilin proteins in the OHC stereocilia disappeared  thresholds like the ABR shifts observed in tubby mice [Tub−/− mice also showed elevated thresholds and reduced amplitudes of their distortion product otoacoustic emissions (DPOAEs), which are used to assess OHC integrity . Sequential mating Tub−/− mice with AKR mice produced offspring that lack Tub but have a wild-type, protective Map1a allele . We found that Tub−/−; Map1aAKR mice show the recovery of ABR threshold shifts and loss of DPOAE amplitudes , suggesting that sensory cells may share pathologic mechanisms related to MAP1A. The detailed roles of MAP1A in the cell surface specializations of photoreceptors and cochlear hair cells are waiting to be discovered. Because there has been no report showing the presence of microtubule in the stereocilia, new function of MAP1A other than the conventional role of stabilizing microtubule can be expected. It is unclear why two seemingly unrelated proteins, MAP1A and tubby, contribute to the proper localization of a single stereociliary molecule. Stereocilia are nano-scale structures. However, they have fine cytoskeletal structures and complex protein interaction networks like primary cilia or neuronal postsynaptic densities. Stereocilin might be a core member of the stereociliary protein interactome whose localization is so important to normal hearing that two mechanisms are necessary. In addition, the independent contributions of MAP1A and tubby to the localization of stereocilin suggest it may be important to consider a “two-hit” mechanism when assessing the roles of stereociliary proteins in hearing impairment.MAP1A plays important roles in stabilizing microtubules in neurons . Howeverent mice . HoweverAdditional file 1: Materials and methods.Additional file 2:Figure S1. Measurements of DPOAE amplitudes. Figure S2. Disappearance of stereocilin from hair cell stereocilia in Tubflox/flox; Pax2-Cre mice.


We administrated fisetin to mdx/utro(−/−) mice for 4 weeks, and observed obviously reduced number of senescent immune cells, restored number of muscle cells, and improve muscle phenotypes. In conclusion, our results reveal that senescent immune cells, such as macrophages, are greatly involved in the development of muscle dystrophy by impacting the function of muscle stem cells, and the senolytic ablation of these senescent cells with fisetin can be an effective therapeutic strategy for improving function of muscle stem cells and phenotypes of dystrophic muscles.The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from  The number of cells positive for β-gal activity at pH 6, a known characteristic of senescent cells, was determined.The presence of senescent cells in skeletal muscle tissue  , fisetin was dissolved in vehicle . Mice were weighed and given fisetin (20 mg/kg for 5 consecutive days each week) or vehicle control alone by oral gavage for 4 weeks.For n = 8) using the RNeasy Mini Kit  according to the manufacturer’s instructions. Reverse transcription was performed using an iScript cDNA Synthesis Kit . The sequences of primers are given in Total RNA was obtained from MPCs or the skeletal muscles of mice (mdx/utr(−/−) mice (n = 8) was visualized by Masson trichrome staining with the Trichrome Stain (Masson) Kit (Sigma-Aldrich). Sections were incubated in Weigert’s iron hematoxylin working solution for 10 min and rinsed under running water for 10 min. Slides were transferred to Biebrich scarlet-acid fuchsin solution for 15 min before incubation in aniline blue solution for another 5 min. Slides were then rinsed, dehydrated, and mounted as earlier. The ratio of the area of fibrotic collagen (blue) to the area of normal muscle (red) was quantified to measure fibrosis formation.Fibrosis formation in muscle tissues of WT or n = 3, with 2 replicates for each cell population) were fixed with 4% paraformaldehyde, and frozen muscle tissue sections were fixed with 10% formalin. The primary antibodies used – CD68 , p-4E-BP1, Pax7 , Collagen IV (Abcam), and fMHC  – were all applied at a 1:100 to 1:300 dilution. Cell nucleus was stained with DNA binding reagent and 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescent images of myofibers were imaged and photographed with a Nikon high resolution microscope.Cultured cells  and Image J software . Data from at least six samples from each subject were pooled for statistical analysis. Results are given as the mean ± SEMx. The statistical significance of any difference was calculated using Student’s


In addition, atypical mitoses were assessed in The Cancer Genome Atlas (TCGA) BC dataset (n = 1032) and were linked to the genetic alterations and pathways. In this study, the median of typical mitoses was 17 per 3 mm2 (range 0–120 mitoses), while the median of atypical mitoses was 4 (range 0–103 mitoses). High atypical mitoses were significantly associated with parameters characteristic of aggressive tumor behavior. The total number of mitoses, and a high atypical-to-typical mitoses ratio (>0.27) were associated with poor BC specific survival (BCSS), . The atypical-to-typical mitoses ratio dichotomized triple negative-BC (TNBC) patients into two distinct groups in terms of the association with the outcome, while the overall number of mitoses was not. Moreover, TNBC patients with high atypical-to-typical mitoses ratio treated with adjuvant chemotherapy were associated with shorter survival (p = 0.003). Transcriptomic analysis of the TCGA-BRCA cohort dichotomized based on atypical mitoses identified 2494 differentially expressed genes. These included genes linked to pathways involved in chromosomal localization and segregation, centrosome assembly, spindle and microtubule formation, regulation of cell cycle and DNA repair. To conclude, the atypical-to-typical mitoses ratio has prognostic value independent of the overall mitotic count in BC patients and could predict the response to chemotherapy in TNBC.Atypical mitosis is considered a feature of malignancy, however, its significance in breast cancer (BC) remains elusive. Here, we aimed to assess the clinical value of atypical mitoses in BC and to explore their underlying molecular features. Atypical and typical mitotic figures were quantified and correlated with clinicopathological variables in a large cohort of primary BC tissue sections ( Visual quantification of mitotic figures in hematoxylin and eosin (H&E) stained BC histological sections, defined as the mitotic score, is the gold standard assessment method that reflects the proliferative status of BC5. In addition, it is an integral component of the Nottingham Grading System6.The proliferative activity of breast cancer (BC) can be considered a surrogate indicator of tumor aggressiveness and provides an important prognostic factor linked to the response to chemotherapy8. Atypical mitosis refers to presence of unusual, dysregulated, and random assembly of nuclear materials within the dividing cells which results in abnormal mitotic morphology which also reflects underlying genomic abnormalities such as chromosomal instability, telomere dysfunction, and aneuploidy10. The presence of atypical mitotic figures is generally acknowledged to be a feature of malignancy and provides prognostic value in certain tumors such as urothelial11 and pancreatic12 carcinomas. Although some studies utilizing a limited number of cases have reported an association between atypical mitoses and poor outcome in BC9, validation studies and characterization of atypical mitoses score in BC remain lacking.Mitotic figures are either typical or atypical14 to relate atypical mitoses to the underlying molecular changes and pathways.In this study, we hypothesized that atypical mitoses provide additional prognostic significance in BC. We have visually quantified atypical mitoses in a large cohort of BC using digitalized whole slide images (WSIs) to assess the relationship between atypical mitoses and patient outcomes. We also used the publicly available RNA sequencing data (RNA-Seq) from The Cancer Genome Atlas (TCGA) BC dataset15 was available  defined as the time (in months) from the date of the primary surgery to the time of death from BC, and distant metastasis‐free survival (DMFS) defined as the time (in months) from the primary surgery until the first event of distant metastasis.A total of 846 primary early-stage BC were included. Detailed clinicopathological data including tumor size, histological tumor grade, histologic type, lymph node status, lymphovascular invasion (LVI), Nottingham Prognostic Index (NPI) and molecular subtypesIn the Nottingham series of cases, we have reviewed 3–4 tumor sections per case, and the representative section, defined as the section with highest tumor grade and burden, was selected to be included in the study. Freshly cut, 4‐μm full‐face H&E sections were prepared from the selected formalin-fixed paraffin-embedded (FFPE) blocks and digitally scanned at 40× magnification using a high‐throughput automated scanner . WSIs were viewed by Case Viewer software  on a full‐screen panel (21-inch screen with 1366 × 768 pixels resolution). Regarding the TCGA cohort, the majority of cases had a single online available H&E digitalized image prepared from FFPE tissue blocks. Few cases had two or more WSIs for the invasive tumor, and the representative section, defined as described above, was selected to be included in this study.2 on digitalized WSI, which is equivalent to 10 high-power fields (HPFs) of the wide field microscope diameter used in routine practice as per our previous study18. Hotspot areas were selected based on being the most cellular area and was usually located at the peripheral invasive front of the tumor19. Initial examination of digital images was carried out at 10× magnification to identify the cellular regions harboring the highest mitotic counts which were quantified at 40× magnification. Areas of fibrosis or necrosis were excluded. Ambiguous structures such as cells with dark hyperchromatic nuclei and/or apoptotic cells were excluded8. Quantifying typical and atypical mitoses was performed separately as the following:(A)20.Typical mitoses were identified by their morphology, in addition to the common features of absence of the nuclear membrane and clearly visible hairy extension of nuclear materials, typical mitosis is characterized by specific arrangement of the chromatin. This includes either clotted (prophase), in plane (metaphase/anaphase), or in separate clots (telophase)(B)21. These include (i) lag atypical mitosis defined as a rod-like structure with one or more unattached chromosome at one side or both sides of metaphase; (ii) spindle multipolarity which shows three attached poles (tripolar), four attached poles (tetrapolar) or more than four poles (multipolar); (iii) dispersed mitosis which is represented by multiple dispersed chromosomes with no specific shapes; (iv) polar asymmetry in which there are two separated masses with unequal size; (v) anaphase bridge that is characterized with a string of chromosome attached to one poles of anaphase; (vi) ring mitosis which is an unusual feature in which chromosomes are displaced to the periphery of the cell8 . Mitotic scores were analyzed with respect to detailed clinicopathological parameters and outcome data. Evaluation of all mitoses was carried out by one observer (AL), who is a well-trained pathologist with more than 5 years’ experience in histopathology and was supervised by a specialized breast pathology consultant (ER). In addition, and to assess the inter-observer concordance, another trained pathologist (MT) scored mitoses in 20% of the study cohort and the agreement between both observers was excellent (kappa = 0.85).Atypical mitoses were defined as mitoses with any morphological appearance other than the typical formsThe overall number of mitoses was evaluated in hotspots within an area of 3 mmn = 1032) 22 was used to study the potential underlying molecular alterations and the differentially expressed genes (DEGs) linked to atypical mitoses. In this cohort, candidate BC histological evaluation was achieved by consensus of a pathology committee13. RNA sequencing was carried out with the Illumina HiSeq platform, and the data was processed using previously described methods23. The resulting sequencing reads were aligned to the human hg19 genome assembly using MapSlice24. RSEM was used to quantify gene expression for the transcript models corresponding to the TCGA GAF 2.13 and was normalized within samples to a fixed upper quartile. Atypical and typical mitoses were counted at 40× magnification per 3 mm2 using the same methodology as described above. DEGs linked with atypical and typical mitoses were identified using the DESeq2 R statistical tool25, used for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. Significant differentially expressed genes were identified based on log2 fold change (≥±1) combined with adjusted p value (<0.05). The web-based gene set enrichment analysis tool  was used to explore significantly enriched pathways and gene ontologies (GO) based on the identified DEGs26. The common DEGs were identified between atypical and typical mitoses using the Venny version 2.0 tool27.TCGA-BRCA cohort (P < 0.05 (two‐tailed) were considered statistically significant.Statistical analysis was carried out using SPSS v.26 . For statistical analysis purposes, atypical mitoses, the overall mitotic counts, and atypical-to-typical mitoses ratio were categorized based on the median, into low and high groups, and were correlated with the clinicopathologic parameters using Chi-square test. The degree of inter-observer agreement in atypical mitoses scoring was assessed statistically using Cohen’s Kappa test. Logistic regression analysis was used to assess the effect of other confounders in the association of atypical mitoses with other variables. The Kaplan–Meier survival curves and log-rank test were used for outcome associations in the whole cohort and in various molecular subtypes. For all tests, 2). The median of typical mitoses was 17 (range 0–120 mitoses), while the median of atypical mitoses was 4 (range 0–103 mitoses). One or more atypical mitoses were present in 77% of the cases  was 21 .2) and other parameters characteristic of aggressive tumor behavior including high tumor grade, larger tumor size, NST histological type, the moderate and poor prognostic NPI groups and (TNBC) phenotype  and DMFS (p = 0.03). However, when the ratio was considered, high atypical-to-typical mitotic ratio showed a strong association with poor outcome (p = 0.013)  Fig. .Fig. 2AsP = 0.001) but not in either TNBC nor in the HER2 subtypes. Similarly, a significant association between high atypical mitoses and both shorter BCSS and DMFS was observed in the luminal BC subtype  , but also in TNBC subtype (p = 0.01)  in the chemotherapy naïve group but not in chemotherapy treated patients . However, when the analysis was carried out on various molecular subtypes, high atypical-to-typical mitoses ratio, showed shorter BCSS in TNBC patients who received chemotherapy (p = 0.003), while this association was not observed when the overall mitoses was considered (p = 0.9)  Fig. .Fig. 3AsP = 0.001). Similar to the previous finding, high atypical mitoses group was significantly associated with parameters characteristic of aggressive behavior like larger tumor size, high tumor grade and TNBC. Similar observation was confirmed regarding high overall mitoses and high ratio.In the TCGA-BRCA cohort, the median of overall mitoses was 18 (range 0–148 mitoses per case) while the median of atypical mitoses was 2 (range 0–97 mitoses). The median of typical mitoses was 13 (range 0–116) and the mean atypical-to-typical mitoses ratio was 0.3 . There was statistically significant difference in the mitoses between the Nottingham and TCGA cohort (n = 3247) that were associated with atypical mitoses, where 2494 genes were significantly associated with high atypical mitoses and 753 genes were associated with low score cases. Regarding typical mitoses, 1864 genes were significantly associated with high typical mitoses whereas 952 genes were associated with low typical mitoses  that can be combined to the other well-known prognostic and predictive factors to add better risk stratification tool in BC as a complementary or even a replacement of the expensive molecular assays. In the era of digital pathology and artificial intelligence, assessment of atypical mitoses and its incorporation within the prognostic and predictive AI-based algorithms is likely to provide a cost-efficient risk assessment tool. Moreover, the actual performance of the available molecular tests is largely similar to the well-established morphological and clinicopathological parameters when assessed accurately using robust methods. Head-to-head comparison of the molecular assays in BC showed similar concordance levels to that reported for histological grade and mitotic scores when assessed in well-fixed samples (Kappa scores = 0.39–0.55), with discordant results being recorded in 41% of patients36. Some studies have reported that chromosomal instability and telomere dysfunction are the main contributors to atypical mitoses in malignancy and that atypical mitosis can be considered a morphological marker of chromosomal instability38. Additionally, It was proposed that presence of atypical mitotic figures is caused by specific centrosomal alterations in each neoplasm, and may be considered as a distinct event that is possibly involved in carcinogenesis39. Aneuploidy is one of the common characteristic features of cancer cells and is related to poor clinical outcomes40. The underlying mechanism for aneuploidy in cancer may be a defect in the mitotic process that is required to segregate duplicated chromosomes into daughter cells41. Atypical mitosis could provide a significant morphological signature of underlying chromosomal instability and aneuploidy of tumor cells.In this study, 77% of the differentially expressed genes (DEG) identified were higher in tumors harboring atypical mitoses and were associated with biological pathways and mechanisms linked to chromosomal localization and segregation, centrosome spindle and microtubule, micro-RNA and DNA breakdown. These finding are consistent with other studies linking atypical mitoses association with genomic instability, telomere dysfunction, chromosomal abnormalities, and aneuploidyThe main limitation of using the atypical-to-typical mitoses ratio in routine practice, is that it depends on independent assessment of both typical and atypical count separately, which may be considered a time-consuming task for pathologists. However, with the emergence of advanced digital pathology and artificial intelligence/machine learn (AI/ML) algorithms, it should be feasible to develop automated image analysis approaches to accurately distinguish and quantify typical and atypical mitoses. In conclusion, our findings suggest that the BC tumors with atypical mitoses possess molecular features associated with an aggressive phenotype, higher risk of metastasis and chemotherapy resistance. Our study supports further investigation of the use of quantitative assessment of atypical mitoses in the context of overall mitotic number as a prognostic indicator in BC.Supplementary material