Passalurusambiguus and Eimeria spp. are two common rabbit intestinal parasites that can cause diarrhoea, among other symptoms, and in severe cases, death. C. guttulatus is a commensal yeast of the rabbits’ stomach that is considered apathogenic but can worsen symptoms in rabbits suffering from coccidiosis. In the present case report, we describe an outbreak of deaths in three different age groups  in an industrial rabbit farm in Greece. Symptoms included depression, diarrhoea, inappetence, weight loss, dehydration and ruffled furs. Using a faecal flotation technique, sick rabbits were found to be moderately to heavily infected with P. ambiguus, Eimeria spp. and C. guttulatus. Treatment with fenbendazole and oregano oil combined with hygiene control measures successfully controlled the infections and resolved clinical symptoms. A faecal flotation method or other reliable diagnostic technique should be used regularly in industrial rabbit farms to screen for gastrointestinal parasitic infections. Early diagnosis and control will help to maintain production levels and, therefore, limit financial losses for the farmer while ensuring animal welfare.Domestic rabbits are commercially farmed for their meat whilst gastrointestinal diseases can hinder their production.  Oryctolagus cuniculus are social animals originating from European rabbits [Passalurus ambiguus [Eimeria spp. [Domestic rabbits belonging to the species  rabbits . Pet rab rabbits , while t rabbits . Commerc rabbits  and fur, rabbits . Rabbit  rabbits . Rabbit  rabbits . One suc rabbits ,7. Indus rabbits . Accordi rabbits . With th rabbits . It is wambiguus  and Eimeria spp. .Passalurus ambiguus , known as the rabbit pinworm, belongs to the Oxyuroidea family and is one of the most common rabbit and hare nematodes with a worldwide distribution [P. ambiguus eggs are ovoid [ribution . It has ribution , embryonribution  with theribution . P. ambire ovoid  and doubre ovoid , which are ovoid  and margre ovoid  and a pore ovoid . Rabbitsre ovoid , or throre ovoid . Adult pre ovoid . Femalesre ovoid . The prere ovoid , and theEimeria spp., which has a direct life cycle. Eimeria spp. parasites have a high host specificity, and rabbits can get infected by at least 14 different Eimeria species. Thirteen of them infect different target sites and depths of the mucosa of the small intestine, caecum or colon, causing intestinal coccidiosis. The last species, Eimeria stiedae, can be found in the endothelial cells of the bile ducts, causing hepatic coccidiosis.Coccidiosis is an ubiquitous  common dEimeria oocysts with their faeces that need to sporulate in order to become infective. Sporulation can take 1–4 days, depending on the species, and is affected by the humidity [Eimeria oocysts, which are not infective [E. stiedai has also been successfully transmitted with blood transfusions from infected to healthy rabbits [Rabbits shed unsporulated humidity  and temphumidity . Oocystshumidity . Transmihumidity . They donfective . Consequ rabbits , intramu rabbits . The pre rabbits  and 14–1 rabbits .Cyniclomyces guttulatus is a budding, sporogenous [Cryptococcus guttulatus and was renamed and reclassified several times before acquiring its final name as Cyniclomyces guttulatus in 1971 [rogenous , symbiotrogenous ,24,25,26rogenous ,24. It w in 1971 .C. guttulatus reaches the uninfected host’s stomach and colonises it if the normal flora allows it [C. guttulatus multiplies rapidly, and the numerous vegetative cells mix with the stomach content. Vegetative cells pass through the gastrointestinal tract and are excreted with the faeces, while some of them form ascospores in the large intestine [Rabbits usually get infected through the faecal-oral route when eating their faeces or other contaminated plants . Followillows it . Cecotrollows it ,24. Aftentestine . Usuallyntestine .An outbreak of deaths was recorded at the start of February 2021 in one of Greece’s largest industrial rabbit farms in the area of Epirus (Ioannina). Rabbits belonged to the HyCole hybrid. The commercial farm consisted of 3000 does in total reared in stainless-steel cages. The farm consisted of different buildings. Each building included six separate rooms with animals of the same age. Cages had a sufficient size allowing rabbits to express normal behaviour. Cage floors were made of wire mesh and cleaned with pressurised water once in two weeks, while the beddings consisted of good-quality hay. Considering as Day 0 the day of kindling, rabbits were bred as follows. Weaning took place on Day 41. Consequently, after Day 75, rabbit slaughtering began and lasted until Day 90–100. All rabbits had ad libitum access to food. Kittens fed on the doe’s milk from Day 0–41. They were also provided with a commercial starter (creep) diet from Day 7–22. Afterwards, from Day 22–52, they were given a grower diet and finally, from Day 52 until slaughtering, they were fed a finisher diet. All does were fed a standard lactation diet. No coccidiostats or anthelmintics were routinely administered to the rabbits.The affected rabbits belonged to both sexes of three different age groups. Group A: lactating does, Group B: 58 days old young rabbits and Group C: 80 days old young rabbits. Each age group was housed in various rooms in separate buildings. Only certain rooms of the buildings included rabbits with clinical signs (most symptoms were common with some variances), while the rest of the rooms housed clinically healthy animals. More specifically, in Group A one room out of six was affected, in Group B one out of six and in Group C two out of six rooms.Group A: lactating does were in their second productive cycle and housed with their 7 days old kittens, which were symptomless. Main clinical symptoms included depression, diarrhoea, inappetence, weight loss, dehydration and ruffled furs. The faeces excreted were dark, soft, watery and foul-smelling. The does experienced the least severe symptoms and recorded only a few deaths compared to other age groups.Group B: 58 days old young rabbits exhibited depression, diarrhoea, inappetence, reduced weight gain, dehydration and ruffled furs. The faeces excreted were dark, soft, watery and foul-smelling.Group C: 80 days old young rabbits exhibited depression, diarrhoea, inappetence, reduced weight gain, dehydration, abdominal distention and ruffled furs. The faeces excreted were dark, soft, watery, foul-smelling, occasionally bloody and mucoid. As a result, rabbits had a dirty perianal area  and a hiAccording to the farm records, the disease was acute, with clinical signs evolving rapidly, resulting in the deaths of the affected rabbits. The farm veterinarian contacted the Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece, and was instructed to provide faecal samples from each group. The samples were collected over 24 h from the animals of all groups and stored in separate urine collection containers until arrival at the Laboratory. They were transported in less than 12 h after collection.We performed a faecal flotation technique on representative pooled samples from 100 animals of each group (from affected rooms). Also, similar pooled samples were examined from 100 healthy animals from each group (originating from rooms that had not any sign of infections). The methodology was as follows. Three grams of faecal material from each container were weighted and placed into separate 15 mL glass conical centrifuge tubes. The tubes were then filled with tap water, and the faecal samples were homogenised with a wooden applicator stick. Faecal samples were filtered through a wire mesh (aperture 250 μm) to eliminate coarse faecal debris, and the faecal suspension was transferred into new tubes. The tubes were then centrifuged at 1500 RPM (Revolutions Per Minute) for 3 min. After centrifuging, the supernatant was discarded, and the tubes were half-filled with a 33% zinc sulfate solution (Specific Gravity: 1.34). The faecal residues were resuspended with a wooden applicator stick, and the tubes were then fully filled with zinc sulfate until a meniscus was formed. An 18 mm x 18 mm square coverslip was carefully placed on top of the tubes before they were centrifuged again at 1000 RPM for 3 min. Subsequently, the coverslips were removed and transferred on separate microscope slides for examination under a light optical microscope  at 100× and 400× magnification. Faecal flotation techniques are not quantitative like the McMaster technique, but they can be used for the semi-quantitative assessment of the number of eggs, oocysts or yeast cells ,30. CritOptical microscopy revealed the following:C. guttulatus vegetative cells and moderate infection (++) with P. ambiguus eggs were found, while in healthy animals from the unaffected rooms only a small number (+) of C. guttulatus vegetative cells was recorded.Group A: In sick animals, heavy infection (+++) with C. guttulatus vegetative cells and heavy infection (+++) with P. ambiguus eggs were found, while in healthy animals only a small number (+) of C. guttulatus vegetative cells was recorded.Group B: In sick animals, heavy infection (+++) with C. guttulatus vegetative cells, moderate infection (++) with P. ambiguus eggs and heavy infection (+++) with different Eimeria spp. oocysts were found, while in healthy animals only a small number (+) of C. guttulatus vegetative cells was recorded.Group C: In sick animals, heavy infection (+++) with C. guttulatus vegetative cells can be seen in P. ambiguus eggs in Eimeria spp. oocysts in Results of the parasitological analysis of affected animals are summarised in P. ambiguus, Eimeria spp. and C. guttulatus, the following treatments were administered to all rabbits from the affected rooms:After identifying P. ambiguus was treated with a commercial suspension containing fenbendazole  administered orally at a dose rate of 20 mg/kg once and repeated in 14 days. No other chemical treatment was applied.Group A: P. ambiguus was treated as in Group A. No other chemical treatment was applied.Group B: P. ambiguus was treated as in Group A. Eimeria spp. was treated with a commercial solution containing oregano  at a dose rate of 500 mL/tonne of drinking water for 7 consecutive days. No other treatment was applied.Group C: C. guttulatus requires the antifungal drug nystatin, and currently, there is no commercial veterinary product in Greece containing nystatin. Furthermore, C. guttulatus is an opportunistic pathogen, and it causes more severe symptoms in rabbits suffering from coccidiosis. On its own, C. guttulatus does not seem to be pathogenic [C. guttulatus was recommended, but a moderate carbohydrate restriction and dietary fibre increase in all animals of the affected rooms. Additional control measures included removing infected faeces and weekly cleaning of cages, particularly cage floors and feed boxes. Facility workers were instructed to wear gloves when handling rabbits and change overalls and shoes between different rooms. Not any treatment was administered to healthy animals housed in the unaffected rooms.The specific products mentioned above are not licensed for use in rabbits. The use of unlicensed drugs is a profound problem for the country’s rabbit industry because most treatments of common diseases require the off-label use of drugs licensed for other animal species. Fenbendazole dose was based on experts’ recommendations in treating passaluriosis ,32. Treathogenic . Based oEimeria spp. oocysts were still present in animals of Group C and only a few C. guttulatus vegetative cells in all rabbits, that were of no clinical importance . The farm veterinarian reported no new diarrhoea cases or deaths. All rabbits tolerated the treatments well, and no side effects were reported. As a result, the farm owner could finally send rabbits from Group C to the abattoir and limit his financial losses.Following treatment, almost all rabbits showed clinical improvement and all the symptoms resolved within 15 days. A follow-up examination of faeces using the same sampling method and flotation technique described above 16 days after treatment revealed no helminth eggs. A small number (+) of P. ambiguus and Eimeria spp. [The present case is a good example of a classic disease in rabbits including three common pathogens , though it has never been linked with deaths of rabbits in Greece. Gastrointestinal disorders that cause diarrhoea in rabbits  can lead to high mortality rates , which iria spp. ,36.P. ambiguus infections can impact production performance in young rabbits and does [P. ambiguus can lead to the parasite’s permanent circulation in industrial rabbit farms if no control measures are taken [P. ambiguus is 55 days, we suggested a faecal examination to be performed on the kittens after weaning, when they reach approximately 67 days of age. Similarly, rabbits in Group B (58 days old) probably acquired the infection from their mothers, and we proposed a faecal examination on those does as well.and does . Auto-inre taken . LactatiMost infections are usually asymptomatic, even with a high parasitic burden . HoweverP. ambiguus [Diagnosis of passaluriosis is achieved with a faecal flotation method, i.e., using a ZnSO4 solution . The traambiguus , and thaIn the case treatment is needed, fenbendazole at a dose of 15–20 mg/kg mixed with feed for five consecutive days has proved successful . PiperazP. ambiguus eggs shed depends on the season and age of rabbits, but not on the sex or spaying of the female rabbits [Infection rates in rabbit farms can reach up to 31% . The num rabbits .Eimeria species are frequently observed [Intestinal coccidiosis is quite common in industrial rabbit farms, especially in young rabbits after weaning. Infections are usually asymptomatic, and mixed infections with different observed , as in oobserved ,41E. flavescens and E. intestinalis, are highly pathogenic, affecting the intestinal crypts. Others are moderately pathogenic, like E. magna and E. media, while some, such as E. perforans and E. coecicola, rarely cause disease [E. perforans has also been linked to intussusception leading to death in a rabbit [E. stiedai infections can also cause ascites or liver and gall-bladder enlargement, jaundice, constipation and fatal liver failure, particularly in young rabbits 60–90 days old [Eimeria species, like in the current case, are not uncommon [E. media, E. magna and E. perforans [Eimeria species they were infected with [Eimeria spp. [Some species, like  disease . As a re disease . Symptom disease . These t disease ,44. In r disease . Additio disease  and mort disease ,46. Cocca rabbit . E. stiedays old . Furtherdays old . All theuncommon ,48,49,50erforans . Howeverted with ,44,51. Iria spp. .Eimeria spp. sizes and characteristics overlap, requiring the use of additional molecular tools.Diagnosis can be achieved by post-mortem examination of the intestinal or hepatic lesions. The different species can be distinguished by their location and lesions caused. Other methods include direct faecal smears, isolation of preferably sporulated oocysts from faeces with a faecal flotation method, the McMaster technique, or other centrifugation techniques ,41. SpecChemoprophylaxis prevents disease occurrence in healthy rabbits while treatment is used to manage disease outbreaks . ChemoprEimeria spp., particularly when administered prophylactically [Salix babylonica [Eimeria spp. However, further research is needed for most of them in order to confirm their effectiveness. Likewise, vaccine development against E. stiedae, E. magna, E. intestinalis and E. media represents a future prospect for the prevention of hepatic and intestinal coccidiosis [Alternative control measures include the administration of non-chemical drugs. There are many herbal formulations, some of which contain oregano, that can be used to control coccidiosis. Oregano’s anticoccidial activity has been demonstrated in broiler chickens . Its effctically ,62,63. Octically ,65 whichctically ,64. The ctically  or with ctically  for coccctically , heterocctically , artemisctically ,71, Salibylonica  and a cobylonica  also shocidiosis ,75,76,77Escherichia coli [Coccidia prevalence in industrial rabbit farms can reach up to 78%  and up thia coli , and othhia coli  in rabbiC. guttulatus, rabbit coccidiosis seems to increase the yeasts’ numbers [C. guttulatus is considered apathogenic, and in the absence of other pathogens, does not cause clinical signs or lesions in rabbits, even in large numbers [C. guttulatus increased their body weight gain and feed intake [C. guttulatus and E. intestinalis had worse symptoms, such as anorexia, reduced body weight, constipation, diarrhoea and mortality, compared to rabbits infected only with E. intestinalis. Besides, rabbits with coinfections shed more vegetative cells but less Eimeria oocysts than rabbits infected only with C. guttulatus or E. intestinalis, respectively [C. guttulatus has also been linked to acute, dark, aqueous, and foul-smelling diarrhoea in rabbits [C. guttulatus vegetative cells which did not have any clinical implications in the absence of other intestinal pathogens.Regarding  numbers . Usually numbers ,26,33,86d intake  and can ectively ,33. C. g rabbits ,87, alonC. guttulatus is considered a pseudoparasite because it closely resembles Eimeria oocysts in faeces and can confuse the diagnostician [Diagnosis can be achieved with a faecal flotation method . The vegostician . In addiostician ,24.In the case treatment is required, it usually involves the oral administration of the antifungal compound nystatin . NystatiC. guttulatus in healthy rabbits, particularly adults. In China, the yeast was found in 83% of rabbits [C. guttulatus infections outside of rabbits [Prevalence can be high for  rabbits . Cases o rabbits ,29,87 ha rabbits  and in d rabbits ,90,91,92P. ambiguus and Eimeria spp. are not uncommon in rabbits. They have been reported in the past, with different prevalence rates, in Greece [P. ambiguus and Eimeria spp. [Mixed infections with n Greece , Germanyn Greece ,94,95, Fn Greece , Turkey n Greece , Poland n Greece , Serbia n Greece , Italy [n Greece , Australn Greece , West Afn Greece  and Kenyn Greece . It is wria spp. . The preE. coli), which were not researched. Diarrhoea in rabbits can also result from dietary or environmental causes, which were outside our study’s aim [P. ambiguus, the deaths ceased, rabbits showed clinical improvement, and no more diarrhoea cases were reported. Thus, we can assume that the diarrhoea and deaths were most likely attributed to those parasites. The large numbers of C. guttulatus may also have worsened the symptoms [We cannot exclude that the deaths in the current case were not associated with other infectious microorganisms , research in ruminants indicates that a smaller sample size should also be sufficient in determining the infestation level . A prereEimeria spp. has also developed some resistance to different coccidiostats because farmers tend to use them for the chemoprevention of coccidiosis in rabbits [Eimeria spp. slowly developed resistance to the specific drug lowering its efficacy [Finally, it is worth mentioning that anthelmintic resistance in gastrointestinal nematodes is on the rise across different animal species worldwide . To comb rabbits . Such isefficacy . In addiefficacy . Likewisefficacy . On the efficacy ,102 and efficacy . Other defficacy . Veterinefficacy .Passalurus ambiguus and Eimeria spp. Sample sizes used for pooling should be based on the expected homogenous or heterogenous distribution of parasites inside each animal group, among other factors. Special attention should be given to the numbers of the otherwise apathogenic yeast Cyniclomyces guttulatus that can aggravate symptoms in rabbits with gastrointestinal disorders. Consequently, if positive results are obtained, strategic treatments, proper hygiene practices, and sanitisation protocols should be employed. Early diagnosis and control will help to maintain production levels and, therefore, limit financial losses for the farmer while ensuring animal welfare.The current study is a good example of a classic disease in rabbits concerning the simultaneous infection with three common pathogens . A faecal flotation method or other reliable diagnostic technique should be used regularly in industrial rabbit farms for the early detection of gastrointestinal parasites, particularly


There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML. This phenotype of LSCs is mainly orchestrated by dysregulated super-enhancers (SEs)/enhancers (Es) and the ensuing dysregulated transcriptome/proteome, which directly results from genetic alterations and/or perturbed levels and activity of epigenetic and transcriptional regulators in AML6. SEs/Es of myeloid lineage transcription factors (TFs) and of their target genes in AML LSCs are characterized by histone H3 lysine (K) 27 acetyl (H3K27Ac) and H3K4 mono-methylation (H3K4Me1) marks, as well as by high occupancy with BRD4 and histone acetyl transferase CBP/p3009. Among the key LSC-specific TFs that regulate cell growth, differentiation and survival of AML stem cell differentiation are RUNX1, PU.1, CEBPα, c-Myb, and c-Myc13. LSD1  is a FAD-dependent amine-oxidase family member and a component of the co-repressor complexes involving HDAC1/2 and CoREST (RCOR1) or NuRD (MTA2)14. LSD1 contains a conserved N-terminal SWIRM domain (100 AA) and C-terminal amine oxidase-like (AOL) catalytic domain15. The AOL domain binds to FAD and the methylated H3K4 substrate, causing demethylation of mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2)16. Two antiparallel α-helices divide and project away from the AOL domain as the Tower domain, which provides the binding interface with CoREST (RCOR1 and HDAC1/2) and allosterically regulates the catalytic activity and stability of LSD116. In addition to H3K4, LSD1 also demethylates TP53, E2F1, and DNMT118. LSD1 interacts with GFI1/1B, a zinc finger transcriptional repressor and master regulator of normal and malignant lineage development and differentiation in hematopoiesis21. GFI1/1B contains an N-terminal, 20 AA SNAG domain and six C-terminal Zn fingers22. LSD1 binds to the methylated lysine in the 8KSKK11 motif in the SNAG (Snail/GFI1) domain, and thereby helps recruit RCOR1-HDAC1/2 co-repressor complex to mediate transcriptional repression and differentiation block due to GFI1 activity in AML stem/progenitor cells22. GFI1/1B can be co-immunoprecipitated with LSD1 and CoREST and exhibits overlapping DNA-binding sites at enhancers  of key myeloid/monocyte differentiation–regulatory genes23. LSD1 inhibitors (LSD1i) disrupt GFI1/1B interaction with LSD1-CoREST, inducing differentiation of AML blast progenitor cells25. Importantly, CRISPR-suppressor scanning revealed that enzymatic activity of LSD1 was not required for blocked AML differentiation and survival26. GFI1/1B interaction with LSD1 and CoREST also causes demethylation of K372 on TP53, thereby inactivating TP5321. Notably, LSD1 is over-expressed in the stem/progenitor versus differentiated sub-types of AML21, and GFI1 expression is a documented prognostic factor in MDS/AML27. LSD1i treatment also increased chromatin accessibility and binding of SPI1 and CEBPα at their target Es/promoters28. Knockdown by shRNA or treatment with either the irreversible tranylcypromine (TCP)-derivative LSD1i or the reversible LSD1i SP2509 disrupted LSD1-binding to CoREST and GFI1/1B, induce differentiation markers (CD86 and CD11b) and morphologic differentiation, repress colony growth, as well as sensitize AML blast progenitor cells (BPCs) to all-trans retinoic acid (ATRA)32. Co-treatment with LSD1i and cytarabine, DNA hypomethylating agents, or inhibitor of HDACs, FLT3, DOT1L or BCL2, was shown to exert synergistic lethality in AML expressing MLL fusion protein30. However, these studies did not interrogate the activity of LSD1i and LSD1i-based combinations in post-MPN sAML blast progenitor cells (BPCs). In present studies, utilizing for the first time CRISPR-Cas9, or LSD1-FKBP12F36V and dTAG-13, we demonstrate that knockout (KO) or degradation of LSD1 inhibits growth and induces differentiation of AML BPCs with or without expression of MLL fusion oncoproteins, and of post-MPN sAML BPCs. While disrupting the binding of LSD1 to CoREST and GFI1, treatment with irreversible LSD1i also attenuated c-Myc levels, but induced expressions of GFI1, PU.1, p21, and CD11b, inhibiting in vitro growth, inducing differentiation/cell lethality, as well as extending survival in immune-depleted mice engrafted with AML or sAML. Utilizing a domain-focused CRISPR-Cas9 sgRNA screen followed by LSD1i treatment, present studies also demonstrate co-dependencies, including BRD4, in AML cells. Whereas co-treatment with an LSD1i and BETi (OTX015) synergistically induced lethality in PD AML including sAML BPCs, pre-treatment with LSD1i sensitized JAKi-resistant post-MPN sAML cells to ruxolitinib and BETi P/R cells to BETi-induced apoptosis33. Co-treatment with LSD1i and BETi (OTX015) also inhibited AML cell burden and improved median survival of NSG mice engrafted with AML or post-MPN sAML cells.During complete remission in AML, AML stem/progenitor cells (LSCs) in the measurable residual disease (MRD) resist differentiation, retain leukemia-initiating potential and mediate relapse of AML34 to degrade LSD1-FKBP12(F36V) in OCI-AML5 cells following knockout of the endogenous LSD1 to assess within hours loss of LSD1 and the downstream biological consequences. As shown in Figs. We first determined effects of CRISPR-Cas9-mediated knockout (KO) of LSD1 on the CoREST complex and on GFI1 and its targets in AML OCI-AML5 and post-MPN sAML SET-2 cells. Fig. 33. Gene set enrichment analyses (GSEA) of the perturbed mRNAs, according to GO and HALLMARK pathways, demonstrated significant positive normalized enrichment scores (NES) for gene-sets, including those of innate immune and inflammatory responses, apoptosis pathways, but negative NES for c-Myc, WNT-β-catenin and translation–initiation pathway genes  and expressing CD11b Fig. . Thus, s37. Co-immunoprecipitation (Co-IP) analyses demonstrated that treatment with INCB reduced binding of LSD1 with CoREST in the AML OCI-AML5, THP1, and in sAML SET-2 cells . Although GFI1-KD blocked INCB-induced GFI1 levels, INCB treatment increased differentiation with greater decline in c-Myc and c-Myb levels   INCB059872 (INCB) and ORY-1001 in AML and post-MPN sAML cellslls Fig. . This walls Fig. A–F25. Toels Fig. . INCB trels Fig. . A simil01) Fig. . These f40. TF motifs in the open chromatin included those of CTCF, FOSL1, PU.1, RUNX1, IRF8 and c-Myc, and the gained peaks included those in GFI1 and PU.1-target genes  or LSD1-KO (via CRISPR-Cas9) on epigenome/transcriptome can only be evaluated several days after gene-editing, we next determined whether these effects also occur soon after a 16-h exposure of AML and post-MPN sAML cells to INCB. Following INCB treatment, ATAC-Seq analysis demonstrated large numbers of lost and gained peaks, including those gained in the bivalent poised enhancers, enhancers, active TSS (transcription start sites) and polycomb-repressed chromatin of OCI-AML5 cells Fig. A, B39,40nes Fig. C–F. Simines Fig. G, H. Altnes Fig. I–K. Notanes Fig. L–N. RNA-nes Fig. A, B. INCnes Fig. C, D. Simnes Fig. . Comparenes Fig. F–K. The 41. Eight days after transduction of gRNAs, cells were treated or untreated with INCB (250 nM) for 4 days. NGS was performed to determine negative selection of the gRNAs identified on day 12 in INCB-treated versus untreated OCI-AML5 cells, compared to the gRNA profile sequenced on day-2 following transduction of gRNAs41. Fig. F36V, which was significantly enhanced by LSD1 degradation following dTAG-13 treatment  Table  targetinent Fig. 10. In thlls Fig. . ChIP-sells Fig. . Consistlls Fig. B, D, E. lls Fig. A, D, E.F43, synergistically induced apoptosis in the indicated AML and sAML cell lines with diverse genetic alterations documented by NGS  and LSD1 depletion by dTAG-13 induced more differentiation, not apoptosis, than either drug-treatment alone . Co-treatment with INCB and WM1119 exerted similar effects as INCB and RGFP966 in the AML and sAML cells   and a DNA hypomethylating agent (decitabine) synergistically induced loss of viability in AML cells  Fig. B–F. Cons.0) Fig. 46. As allls Fig. B, C25.p < 0.005). Co-treatment with INCB and ruxolitinib also synergistically induced lethality in HEL92.1.7 and SET-2, as well as in samples of PD post-MPN sAML blasts in which genetic mutations were documented by NGS    to ruxolitinib-induced loss of cell viability . In sAML#20, CRISPR-Cas9-mediated LSD1-KO depleted protein levels of LSD1, CoREST and c-Myc, but increased GFI1, GATA2, CEBPα, p21, and CD11b, without altering protein levels of HDACs1/2   AML blasts with genetic alterations documented by NGS Fig. . We dete0) Figs.  and S12D01) Fig. 47. Pre-tity Fig. . Additio1/2 Fig. . These s.0) Fig. .Fig. 5Co33. Exposure to INCB for 96 h dose-dependently induced differentiation and apoptosis to a similar level in SET-2 and SET-2-OTX P/R as well as HEL92.1.7 and HEL-OTX P/R cells, although exposure to INCB for 48 h was relatively inactive in inducing apoptosis  post-MPN sAML and AML cells  that displayed nongenetic mechanism of BETi-resistance, based on increased expression and activity of β-catenin-TCF7L2-JMJD6-c-Myc Fig. 33. Exposis Figs. . Howeveris Figs. . Pretreais Figs. C, D. In is Figs. . Westernis Figs. . A signines Fig. A–C. Consnes Fig. D–G. ThesGiven the marked in vitro synergy noted above, we determined in vivo antileukemia efficacy of INCB alone or co-treatment with ruxolitinib or OTX015 in NSG mice engrafted with AML or post-MPN sAML cells. Treatment with INCB alone for 3–4 weeks reduced AML and sAML burden and significantly improved overall survival of NSG mice engrafted with OCI-AML5 or HEL92.1.7 cells, respectively Figs. A, B. Add50. CRISPR-suppressor scanning was utilized to show that LSD1 enzyme activity is not required for AML survival26. Instead, LSD1 inhibitors disrupt interaction between LSD1 and transcription repressor GFI1/1B on the chromatin. Also, in a knockdown and rescue experiment, the LSD1 K661A catalytic mutant was as capable of rescuing the clonogenic potential of LSD1 knockdown cells as the wild type LSD1 protein50. Additionally, LSD1 inhibitor treatment induces rapid and extensive transcriptional alterations, which do not correlate with LSD1i-mediated changes in H3K4 mono- and dimethyl-chromatin marks50.Utilizing the dTAG system to degrade LSD1 and CRISPR-Cas9 to edit and KO LSD1, findings presented here highlight gene-expression perturbations that lead to differentiation of AML and post-MPN sAML blasts. Also, disruption of LSD1 interactions with CoREST-HDAC1/2 complex and with GFI1/1B by LSD1i activates GFI1/1B-repressed enhancers and transcription factors, including PU.1, CEBPα and IRF8 while repressing c-Myc, and their target gene-expressions. This induces cell cycle inhibitors including p21 and p27, up-regulates differentiation-associated gene-expressions such as CD11b, CD86, LY96, and LYZ, and induces morphologic features of AML cell differentiation. Although LSD1i inhibits both the scaffold and enzymatic functions of LSD1, AML cell-differentiation occurs independent of inhibiting enzymatic demethylase function of LSD147, which could serve as a predictive early biomarker of achieving biologically effective intracellular levels of LSD1i in AML and post-MPN sAML cells. LSD1-KO also increased H3K27Ac and BRD4 occupancy at the GFI1 locus inducing GFI1 in AML cells. However, it is noteworthy that without intact LSD1-CoREST-HDAC1/2 complex, upregulated GFI1/1B is unable to repress differentiation in AML/sAML cells21. Studies presented here also involved CRISPR-Cas9-mediated KO of LSD1 or shRNA-mediated KD of LSD1, which showed induction of GFI1/1B, as well as of PU.1, CEBPα, and p21, but repression of c-Myc in both post-MPN-sAML and non-MPN associated AML cells. Notably, reduced H3K27Ac occupancy at the MYC, but increased at the ITGAM, LY96, and LYZ loci, induced ITGAM, LY96, and LYZ expressions, differentiation and cell lethality of AML and post-MPN sAML blasts. Our findings also confirmed that CRISPR-Cas9-mediated GFI1-KO or shRNA-mediated GFI1-KD also induced differentiation markers and morphologic features of differentiation in AML cells, albeit to a lesser extent than knockout or knockdown of LSD1. However, these findings also demonstrated that LSD1i treatment augmented differentiation due to GFI1-KD. This suggests that disruption of LSD1 binding to repressive complexes not involving GFI1/1B may also be contributing to LSD1i-induced differentiation in AML cells51. Alternatively, it also suggests that both scaffold and enzymatic functions of LSD1 (inhibited by INCB) may be involved in AML cell differentiation induced by INCB38. Again, LSD1i-induced differentiation was also associated with induction of GFI1, PU.1, and p21, but depletion of c-Myc in both post-MPN-sAML and non-MPN associated AML cells. This highlights targeted degradation and depletion of LSD1, which would disrupt both scaffolding and enzymatic activities of LSD1, as an attractive therapeutic approach. Notably, LSD1-KO decreased H3K27Ac occupancy at enhancers/promoters and mRNA expressions of MYC and WNT-β-catenin-target genes, associated with positive enrichment of HALLMARK gene-sets of innate immunity, inflammatory response and apoptosis in AML cells. LSD1-KD by shRNA also positively enriched similar HALLMARK gene-sets in AML cells. These findings are consistent with recent reports that LSD1i treatment stimulates antitumor immunity by inhibiting Foxp3+ Treg cell function, thus enabling and enhancing immune checkpoint blockade53. Collectively, these findings highlight testing potential utility of including LSD1i co-treatment with immune therapies in AML.Additional findings highlighted here are that disruption of LSD1-CoREST-HDAC1/2 complex derepresses GFI1/1B SE/Es to cause early induction of GFI1/1B56. Synergistic in vitro lethality was also observed due to co-treatment with LSD1i and agents targeting HDAC3, MOZ or DOT1L. However, whether these LSD1i-based combinations would also exert superior in vivo anti-AML or anti-sAML efficacy remains to be determined. Notably, a dual class I HDACi and LSD1 inhibitor was shown to exert synergistic lethality in AML as well as in tumor models other than AML58. CRISPR-Cas9 screens in AML cells expressing MLL-fusion gene showed superior efficacy of co-targeting mTORC1 with LSD1, which was not revealed by our screen59. This difference is because our domain-focused CRISPR-Cas9 screen involved AML cells without MLL fusion gene-expression, and utilized gRNAs targeting chromatin regulators not mTORC1. Our findings also demonstrate that co-treatment with LSD1i and the JAK1/2 inhibitor ruxolitinib exerts synergistic in vitro lethality and exhibited superior in vivo efficacy than LSD1i monotherapy in a mouse xenograft model of post-MPN sAML. A recent report highlighted that LSD1i treatment exhibited clinical efficacy by reducing spleen size and constitutional symptoms in patients with advanced MPN, thus combination therapy with LSD1i and JAKi merits further in vivo evaluation60. Our studies demonstrating synergistic in vitro lethality due to combination of LSD1i with decitabine also merits further verification through in vivo AML xenograft studies.Utilizing a protein domain-focused CRISPR-Cas9 screen, our studies revealed that BRD4, DOT1L, HDAC3 and MOZ could serve as effective co-targets to achieve synergistic efficacy with LSD1i against AML and sAML cells. Consistent with this, LSD1i and BETi co-treatment exhibited synergistic loss of viability in PD AML and post-MPN sAML blasts displaying diverse genetic alterations. In addition to abrogating LSD1i-induced GFI1 and PU.1, BRD4 inhibition by BETi increased lethal activity of LSD1i most likely due to observed greater decline in c-Myc and increased p21 and p27 protein levels. Additionally, in previous reports, we have highlighted that inclusion of BETi enhances lethal activity of BETi-based combinations due to greater depletion of c-Myc, cell cycle dependent kinases CDK4 and CDK6, as well as concomitant induction of CDK inhibitor p21, pro-apoptotic (BIM) and downregulation of anti-apoptotic  proteins.63. Agents that target signaling kinases  and epigenetic mechanisms  were shown to result in enhancer reprogramming via newly-marshaled activities of lineage-specific transcriptional regulators which creates the transcriptome/proteome conferring drug tolerant persister/resistance in AML cells49. We previously reported that increased c-Myc expression due to increased levels and activity of β-catenin-TCF7L2-JMJD6-MYC axis induced nongenetic BETi-persister/resistance in AML and sAML blasts33. This was due to enhancer reprogramming and increased transcriptional activities of MYC, RUNX1, and TCF7L2, based on H3K27Ac and BRD4 occupancy at their Es/promoters33. Our findings demonstrate, for the first time, that LSD1i is as active in inducing differentiation and lethality in the BETi-P/R as in BETi-sensitive sAML cells. Notably, sensitization of BETi-P/R sAML and AML cells to BETi-induced apoptosis by pre-treatment with LSD1i was associated with increased chromatin accessibility and mRNA expressions of GFI1, PU.1, and IRF8-target genes. However, mRNA expressions of c-Myc targets were mostly downregulated. Importantly, our findings also show that LSD1i pre-treatment similarly sensitized sAML-RuxP cells that display nongenetic resistance to ruxolitinib48. Taken together, these findings highlight that, by reprogramming of SEs/Es and modifying gene-expressions involved in conferring nongenetic persister/resistance, LSD1i treatment could sensitize AML to BETi, and sAML cells to ruxolitinib or BETi, thereby highlighting novel differentiation and lethal therapies for AML or post-MPN sAML. Findings presented here also beg the question where in AML or post-MPN sAML therapy LSD1i-based combinations should best be clinically tested for their efficacy, i.e., during induction therapy or during consolidation therapy to effectively eliminate the MRD. Due to their demonstrable ability to induce differentiation and eliminate AML blast progenitors, as well as in augmenting immune checkpoint blockade therapy, LSD1i-based combinations should be likely best tested for efficacy in patients with AML in CR but detectable MRD that eventually relapses as therapy-refractory AML.There is a growing evidence and recognition of epigenetic heterogeneity and plasticity in cancer cells, which under selection pressure of targeted therapies facilitates emergence of drug-tolerant persister/resistant cancer cells displaying nongenetic therapy-resistanceOCI-AML5, OCI-AML2, Mono-Mac-1, MOLM13, SKM-1, and SET-2 cells cells were obtained from the DSMZ. MV4-11, HEL92.1.7, THP1, and HS5 cells were obtained from the ATCC . HEK-293T cells were obtained from the Characterized Cell Line Core (CCLC) at M.D. Anderson Cancer Center, Houston TX. Experiments with cell lines were performed within 6 months after thawing or obtaining from ATCC or DSMZ. Cell lines were authenticated in the CCLC at M.D. Anderson Cancer Center. Logarithmically growing, mycoplasma-negative cells were utilized for all experiments.Untreated or drug-treated cells were stained with Annexin-V  and TO-PRO-3 iodide  and the % of apoptotic cells were determined by flow cytometry. To analyze synergism, cells were treated with combinations for 48 h and the % of apoptotic or nonviable cells were determined by flow cytometry. The combination index (CI) for each drug combination was calculated utilizing CompuSyn software. We also utilized matrix dosing of agents in combinations to allow synergy assessment by Bliss scores utilizing the SynergyFinder online web application tool.Following the designated treatments, total RNA was isolated from AML or sAML cells utilizing a PureLink RNA Mini kit from Ambion, Inc.  and reverse transcribed. Quantitative real time PCR analysis was performed on cDNA using TaqMan probes. Relative mRNA expression was normalized to GAPDH and compared to the untreated cells.t-test. For the in vivo mouse models, a two-tailed, unpaired t-test was utilized for comparing total bioluminescent flux. For survival analysis, a Kaplan–Meier plot and a Mantel–Cox log rank test were utilized for comparisons of different cohorts. P values of <0.05 were assigned significance.Significant differences between AML or sAML cells treated with different experimental conditions compared to control cells were determined using the two-tailed, unpaired Reproducibility ChecklistSupplemental Figure legendsSupplemental FiguresSupplemental Materials and MethodsTable S1. Guide RNA targets and sequences


GGCX gene has been associated with asthenozoospermia. In addition, bioinformatic analysis also suggests the involvement of a vitamin B6-axis in calcium-dependent MGP-mediated protein aggregation. These findings suggest that vitamins interact with calcium homeostasis in the epididymis to ensure proper sperm maturation and male fertility. This review article discusses the regulation mechanisms of calcium homeostasis in the epididymis, and the potential role of vitamin interactions on epididymal calcium homeostasis, especially the role of matrix calcium in the epididymal lumen as a cofactor for the carboxylated MGP-mediated scavenging function.In the male reproductive tract, the epididymis is an essential organ for sperm maturation, in which sperm cells acquire mobility and the ability to fertilize oocytes while being stored in a protective microenvironment. Epididymal function involves a specialized luminal microenvironment established by the epithelial cells of epididymal mucosa. Low-calcium concentration is a unique feature of this epididymal luminal microenvironment, its relevance and regulation are, however, incompletely understood. In the rat epididymis, the vitamin D-related calcium-dependent TRPV6-TMEM16A channel-coupler has been shown to be involved in fluid transport, and, in a spatially complementary manner, vitamin K2-related γ-glutamyl carboxylase (GGCX)-dependent carboxylation of matrix Gla protein (MGP) plays an essential role in promoting calcium-dependent protein aggregation. An SNP in the human  The production of viable and functionally competent spermatozoa is a prerequisite for male fertility. This is achieved through normal spermatogenesis in the testis and maturation of spermatozoa in the epididymis. Spermatozoa are non-fertilising when released from the testis but become functionally competent during epididymal transit. The fertilizing competence of spermatozoa is conferred by their interaction with the epididymal luminal microenvironment, which is formed by the epithelial cellular activities and retained behind the blood-epididymis barrier . SpermatEpididymal function relies on a highly specialized epididymal luminal microenvironment, which is formed and maintained by the well synchronized cellular activities of the epithelial cells lining the epididymal mucosa. Owing to the complex functions and compartmentalisation of the epididymis, multiple causes of epididymal dysfunction resulting in male fertility disorders, and even the health of offspring, are conceivable . Defects2+) concentration -mediated scavenging of extracellular metabolites in the epididymal microenvironment will be discussed, and the potential role of vitamins in this regard will be explored.As in other systems, Ca network . In thisWhereas the essential roles of vitamins in general biology are well recognized, their specific roles in male reproductive health are remained incompletely understood. It has been reported that specific vitamin supplements can increase sperm quality in rats , demonst2+ homeostasis pathways. For example, vitamin B6 deficiency in rats was found to alter intracellular Ca2+-homeostasis in enterocytes, potentially via Ca2+ channel modulation without affecting net Ca2+ transport - and transmembrane protein 16A -associated activities in Ca2+- and fluid-homeostasis  signaling axis from the basolateral side   , aquaporr (CFTR) , and thesporters , have imsporters .Water is the medium of numerous hydrolysis processes in biological systems and carbon dioxide and its hydrolyzed derivative bicarbonate are not only the metabolic products but also an acid-base balancers in the body. In epididymis, bicarbonate-related acid-base balance is an essential process for acidification of luminal fluid, which is critical for maintaining spermatozoa in a quiescent state . Hence, + ion concentration, up to approximately 55 mM in the distal cauda region (approximately ten-fold higher than in serum), and the low concentration of Ca2+  are notable. These special properties indicate that the microenvironment for sperm maturation is uniquely formed and maintained as spermatozoa transit the epididymal tubule.2+ in the cauda depend on active Ca2+ reabsorption machinery in the epididymis. Consistent with the critical role of a low Ca2+ concentration in the epididymal fluid, dysregulation of epididymal Ca2+ homeostasis is one cause of male infertility , which operate against the electrochemical gradient for Ca2+ absorption: Ca2+-selective ion channels TRPV5 and TRPV6  knockout mice, sperm lacked motility and could not move forward in the female reproductive tract, resulting in male infertility , all of these physiological features are uniquely formed and maintained in the epididymis. Therefore, the coupling of TRPV6 with TMEM16A makes this coupler a key player of epididymal secretion. TMEM16A has been described to mediate epithelial mucin secretion, including in response to inflammation -mediated carboxylation of the substrate MGP to play a role in Ca2+ homeostasis in the epididymis  residues. It was first described as an extrahepatic matrix protein in the extracellular matrix of bone and cartilage and later detected in a wide variety of other tissues such as lung, heart, kidney and arterial vessel walls  in the GGCX protein , rs699664, in the GGCX gene of infertile men with asthenozoospermia . This SN protein . The sam protein . The SNP protein . This prproteins .325 residue of GGCX is responsible for stabilizing the negatively charged Glu residue of MGP during the stepwise carboxylation process, which is promoted in the acidic conditions of the epididymal lumen. Such charge-charge interaction stability is lost as a result of the SNP, in which the charged arginine325 residue is replaced with the neutral glutamine325, thereby changing the binding preference of MGP. Accordingly, the charge-charge interaction stability in the wild-type protein ensures sufficient time for complete carboxylation of each residue in the substrates and avoids their incomplete activation (2+-binding, disturbed Ca2+-homeostasis, and ultimately to impaired sperm maturation and to male infertility.While further investigation is required, we speculate that the positively charged argininetivation . Supporttivation . We spec2+ homeostasis, including fat- or water-soluble vitamins, as illustrated in 2+ homeostasis can be antagonized by vitamin A, resulting in higher incidents of osteoporosis in man (2+ homeostasis in a spatial complementary manner (2+-bound carboxylation-dependent MGP in the epididymal Ca2+, we performed a bioinformatic analysis on our published proteome results (2+-dependent MGP-aggregation, as demonstrated in 2+ homeostasis.An interplay of multiple vitamins may be required for optimal Cas in man . A syners in man . Consisty manner . To eluc results , and we 2+-bound MGP. In addition, we found that phagosome and antigen processing and presentation as the other key enriched pathways, which were separated from the main primary cluster of metabolic pathways. This may suggest that bound MGP-dependent Ca2+-chelation participates in immunological function in the epididymis, which is essential for sperm function (2+-MGP aggregates. The metabolite products of the identified pathways are substrates for oxidative phosphorylation and fatty acid elongation. These results suggested that Ca2+-bound MGP aggregates are involved in lipid generation, together with the carbohydrate-related catabolism. In view of the maturing spermatozoa in the epididymal lumen, and the various vitamin-dependent pathways in Ca2+-homeostatic regulation, we hypothesized that there is an interplay of different vitamins in the regulation of Ca2+ homeostasis in the epididymis, and that the biphasic chelation activity of carboxylated MGP contributes to scavenging of extracellular metabolites, whereas Ca2+ serves as the cofactor in this process.On the basis of the pathway network analysis in function . The dow2+ homeostasis in maintaining bone health and for preventing vascular ectopic calcification (2+ signaling (2+ homeostasis mechanisms in the epididymis should provide insights into the causes of epididymal-dependent sperm dysfunction and male infertility.It is known that vitamin K is a key modulator of Cafication , which mfication . Since tignaling , the elu2+ homeostasis, particularly extracellular Ca2+ homeostasis in epididymal fluid. Therefore, low Ca2+ is a key factor in regulation of sperm maturation and in male reproductive health. Taken together, the evidence presented here indicates that dysregulated Ca2+ homeostasis due to defective expression of Ca2+-binding proteins, abnormal Ca2+ signaling, or different kinds of vitamin deficiency in the male genital tract may negatively affect male reproductive health.While further investigations are required for a full picture of the interplay between the vitamin signaling pathways, all pathways are connected to Ca2+homeostasis of acidity, both of which are strictly regulated by a network of cell-cell interactions and signaling pathways, as depicted in 2+ in functioning as a cofactor for the GGCX-dependent carboxylation of MGP, which in turn acts as a scavenger for extracellular metabolites. In addition, we discussed potential interactions of vitamins in these pathways, which are essential for sperm maturation and male reproductive health. This raises the possibility that these multi-vitamin-dependent Ca2+ homeostatic-dependent pathways could be leveraged for novel interventions aimed at treating and preventing sperm dysfunctions and male reproductive defecits.Accumulating evidence has indicated the important role of epididymal function in male fertility outcomes, and this function depends to a large degree on the congenial luminal microenvironment established by the epithelial cells lining within the excurrent duct. Important aspects of the luminal microenvironment include Ca


Channel activation is initiated by reactive oxygen species (ROS), leading to a subsequent increase in ADP-ribose and the binding of ADP-ribose to an allosteric site in the cytosolic NUDT9 homology domain. In many animal cell types, Ca2+ entry via TRPM2 channels mediates ROS-initiated cell injury and death. The aim of this review is to summarise the current knowledge of the roles of TRPM2 and Ca2+ in the initiation and progression of chronic liver diseases and acute liver injury. Studies to date provide evidence that TRPM2-mediated Ca2+ entry contributes to drug-induced liver toxicity, ischemia–reperfusion injury, and the progression of non-alcoholic fatty liver disease to cirrhosis, fibrosis, and hepatocellular carcinoma. Of particular current interest are the steps involved in the activation of TRPM2 in hepatocytes following an increase in ROS, the downstream pathways activated by the resultant increase in intracellular Ca2+, and the chronology of these events. An apparent contradiction exists between these roles of TRPM2 and the role identified for ROS-activated TRPM2 in heart muscle and in some other cell types in promoting Ca2+-activated mitochondrial ATP synthesis and cell survival. Inhibition of TRPM2 by curcumin and other “natural” compounds offers an attractive strategy for inhibiting ROS-induced liver cell injury. In conclusion, while it has been established that ROS-initiated activation of TRPM2 contributes to both acute and chronic liver injury, considerable further research is needed to elucidate the mechanisms involved, and the conditions under which pharmacological inhibition of TRPM2 can be an effective clinical strategy to reduce ROS-initiated liver injury.TRPM2 channels admit Ca The liver is central to whole body metabolism and to the co-ordination of metabolic pathways for carbohydrate, lipid, protein, and amino acid metabolism (reviewed in ). In addMany acute and chronic liver diseases are recognised. For almost all of these, oxidative stress and reactive oxygen species (ROS) play a significant role in the initiation and progression of the disease ,6,7,8,9.2). The chronic diseases or acute injuries listed above provide intracellular conditions favourable for increased activity of the reactions that generate superoxide and/or a rapid increase in O2, such as what occurs in ischemia–reperfusion cyt) ,10,13,142+-permeable channels in the plasma membrane are known to be activated by ROS cyt is proposed to amplify the physiological intracellular Ca2+ signals cyt, and pharmacological inhibitors  is widely used to treat many types of pain, and if used at the recommended dose has few side effects ,62. Howe2+]cyt, culminating in necrotic cell death cyt initiated by acetaminophen overdose appears to play a central role in the initiation and progression of necrosis . Ca. Ca2+ en2O2, is relatively slow in onset [2O2 or acetaminophen induced co-localisation of TRPM2 and cadherin at the plasma membrane .2+ entry into hepatocytes. Badr and colleagues investigated the roles of several TRP channels known to be activated by ROS in mediating acetaminophen-initiated increases in [Ca2+]cyt in immortalised human HepG2 liver cells [2+ imaging was employed to assess Ca2+ entry via increases in [Ca2+]cyt, and siRNA directed against a given TRP channel and pharmacological inhibitors were used to identify the channels activated by acetaminophen. It was concluded that treatment of HepG2 cells with acetaminophen or H2O2 activates Ca2+ entry through the TRPV1, TRPC1, TRPM2, and TRPM7 channels, with TRPV1 and TRPC1 being the major contributors cyt, Ca2+/calmodulin-dependent protein kinase II phosphorylation, and expression of mRNAs encoding IL-1beta and IL-6. These palmitate-induced changes were all reduced by saliroside. The authors concluded that the observed actions of saliroside are due to inhibition of expression of TRPM2 and subsequent reduction in [Ca2+]cyt, leading to decreased activation of Ca2+/calmodulin-dependent protein kinase II, increased autophagy, and decreased inflammation cyt, curcumin reduces ROS through mechanisms independent of TRPM2 channels, as discussed above cyt plays an important role in causing hepatocyte injury and death in drug-induced liver toxicity, and likely also in ischemia–reperfusion injury and the progression of non-alcoholic steatohepatitis to cirrhosis and hepatocellular carcinoma. Given the importance of ROS in liver diseases and of TRPM2 and intracellular Ca2+ in mediating ROS-induced cell injury and death in other cell types and organs, it is somewhat surprising that there have so far been relatively few studies of the role of TRPM2 in oxidative stress in liver diseases. Further research, directed towards better definition of the nature of the downstream pathways and organelles affected by TRPM2-mediated increases in [Ca2+]cyt, and the time courses involved, will be invaluable. As will further investigation of the balance between the detrimental and beneficial pathways initiated by TRPM2 channels in ROS-mediated liver diseases.Functional TRPM2 channels are expressed in the liver in hepatocytes and likely also in Kupffer cells. They mediate CaPharmacological inhibition of TRPM2 offers a strategy to reduce ROS-mediated liver injury, but this approach is currently hampered by lack of a suitable TRPM2 inhibitor with high affinity and specificity that can be delivered clinically with reasonable bioavailability. One candidate for such a potential TRPM2 inhibitor is the natural product curcumin. Apart from its acceptance as being safe for the treatment of human diseases, curcumin has multiple actions, especially non-enzymatic removal of ROS, in addition to inhibition of TRPM2 channels. However, considerable further studies are needed to define the mechanism by which curcumin inhibits TRPM2 and to test its potential clinical usefulness for the treatment of liver diseases.


The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca Inflammation is a component in common to both allergic conjunctivitis and dry eye disease – two frequently occurring diseases of the ocular surface (cornea and conjunctiva) . In alleConjunctival goblet cells synthesize and secrete large, high molecular weight, gel forming mucins . These m2 activity, thus blocking the production of pro-inflammatory eicosanoids  to the required concentration and added to the conjunctival goblet cells. RPMI-1640 cell culture medium, penicillin/streptomycin, trypsin, and L-glutamine were purchased from Lonza . Fetal bovine serum (FBS) was from Atlanta Biologicals . Fura-2 was from Life Technologies . U73122 and U73343, KN92 and KN93 were purchased from Tocris Bioscience . The lectin Ulex Europaeus Agglutinin (UEA-1), histamine, BAPTA/AM, 2-aminoethyl diphenylborinate (2-APB), 1-butanol (1-but), t-butanol (t-but), carbachol, aristolochic acid (AA), thapsigargin and RO317549 were all purchased from Sigma-Aldrich . UO126 was obtained from R&D Systems . H89 and synthetic lipoxin A4 ,6(R),15(S)-TriHETE) were from EMD Millipore . Resolvin (Rv) D1  was purchased from Cayman Chemical, Ann Arbor, MI). N-Boc-Phe-Leu-Phe-Leu-Phe (BOC2) was obtained from Genscript Corp in Piscataway, NJ.AnxA1 was obtained from MyBiosource . The compound was stored at -20°C. Prior to use, AnxA1 was immediately diluted in Krebs-Ringer bicarbonate buffer with HEPES  were anesthetized with CO2+]i measurements or mucin secretion measurements, respectively. First passage goblet cells were used in all experiments. The identity of the cultured cells was checked by evaluating staining with antibody to cytokeratin 7 (detects goblet cell bodies) and the lectin UEA-1 (detects goblet cell secretory products) to ensure that goblet cells predominated in the cultures.Goblet cells from rat conjunctiva were grown in organ culture as described previously . The conCultured goblet cells were homogenized in TRIzol and total RNA isolated according to manufacturer’s instructions. One microgram of total RNA was used for complementary DNA (cDNA) synthesis using the Superscript First-Strand Synthesis system for RT-PCR . The cDNA was amplified by the polymerase chain reaction (PCR) using primers specific to AnxA1 using the Jumpstart REDTaq Readymix Reaction Mix  in a thermal cycler . The forward primer was GTG ATC GCT GTG AGG ATA TGA G, and the reverse primer was TAC AGA GCA GTT GGG ATG TTT AG. These primers generated 504 bp fragments. β-Actin primers, the positive control, were forward CGT CAT ACT CCT GCT TGC TGA TCC A and the reverse primer was ATC TGG CAC CAC ACC TTC TAC AAT GG CT and generated a 790 bp fragment. The conditions were as follows: 5 min at 95°C followed by 35 cycles of 1 min at 94°C, 30 seconds at annealing temperature for 1 min at 72°C with a final hold at 72°C for 10 min. Samples with no cDNA served as the negative control. Amplification products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.Cultured rat goblet cells were plated in 24-well plates and grown to confluence , 18, 19.2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 MM NaHCO3, 10 mM HEPES, and 5.5 mM glucose) (pH 7.45) plus 0.5% BSA containing 0.5 fura-2/AM , 8 μM pluronic acid F127  and 250 μM sulfinpyrazone (Sigma-Aldrich), followed by washing in KRB-HEPES containing sulfinpyrazone. Ca2+ measurements were made with a ratio imaging system  using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. A minimum of 10 cells were selected in each experimental condition. AnxA1 was added either alone or after incubation with inhibitors for 15 or 30 min and intracellular [Ca2+] ([Ca2+]i) measured. After addition of agonist, data were collected in real time. The data are presented as [Ca2+]i over time and peak [Ca2+]i, calculated by subtracting the average value before the agonist AnxA1 was added  from the peak [Ca2+]i.Cultured rat conjunctival goblet cells were transferred to 35-mm glass bottom culture dishes and incubated overnight. The goblet cells were then incubated for 1 h at 37°C with Krebs-Ringer bicarbonate buffer with HEPES (KRB-HEPES)  or a positive control histamine (10-5 M)  such as LXAet cells –12. To e(10-5 M) , 23 and (10-5 M) Figure 2(10-5 M) , 24. Anx2+]i. Cultured goblet cells were incubated with fura-2/AM before stimulation with AnxA1 (10−11 -10−8 M). AnxA1 increased [Ca2+]i in a time and concentration-dependent manner  for 30 minutes prior to AnxA1 (10-9 M) stimulation. The cholinergic agonist carbachol (Cch) at 10-4 M was used as a control. To ensure that BAPTA chelated [Ca2+]i and prevented the increase in [Ca2+]i induced by AnxA1 and Cch, [Ca2+]i was measured , stimulated by AnxA1 (10-10 M) and secretion measured. In three independent experiments, AnxA1 alone significantly increased glycoconjugate secretion by 1.5 ± 0.05 fold above basal (-4 M)-stimulated glycoconjugate secretion was also significantly inhibited from 1.7 ± 0.2 fold to 1.1 ± 0.1 fold above basal. These findings demonstrate that AnxA1 increases [Ca2+]i to stimulate mucin secretion in conjunctival goblet cells.We then investigated if AnxA1 uses 2+ stores play in AnxA1 stimulation, goblet cells were incubated with the Ca2+ reuptake inhibitor thapsigargin. Thapsigargin depletes the ER of Ca2+ by blocking the Ca2+/ATPase in the endoplasmic reticulum (ER) that inhibits Ca2+ from being taken up by the ER  added to the KRB containing CaCl2 significantly increased [Ca2+]i by 124.1 ± 23.0 nM , a control, elevated [Ca2+]i to 160.4 ± 40.9 nM. Cch-stimulated increase in [Ca2+]i was significantly blocked by the CaCl2-free solution and was 11.8 ± 2.1 nM. Based on these results, AnxA1 releases Ca2+ from intracellular Ca2+ stores and stimulates extracellular Ca2+ influx to increase the [Ca2+]i in conjunctival goblet cells.After an agonist depletes intracellular Ca-4 M) for 30 minutes before stimulating the goblet cells with AnxA1 (10-9 M) or LXA4 (10-9 M), which is known to bind to ALX/FPR2 and is a positive control  , 26. To  control . AnxA1 a-4 M) followed by AnxA1 (10-10 M) (4 (10-9 M) was again used as a positive control. Secretion was significantly increased by 1.5 ± 0.05 fold above basal when AnxA1 (10-10 M) was added by itself. AnxA1 stimulated secretion was significantly inhibited by BOC-2 to 0.74 ± 0.16 fold above basal. LXA4 stimulated secretion was also inhibited by BOC-2. These data indicate that AnxA1 uses the ALX/FPR2 receptor to stimulate an increase in [Ca2+]i and secretion in conjunctival goblet cells.To determine if AnxA1 uses the ALX/FPR2 receptor to stimulate high molecular weight glycoprotein secretion, cells were preincubated with BOC-2  and diacylglycerol (DAG) . To explore if AnxA1 activates the PLC pathway in rat conjunctival goblet cells, these cells were preincubated with either the PLC inhibitor U73122 or its negative control U73343 at 10-7 M, before addition of AnxA1 (10-9 M) or a positive control Cch [10-4 M)  pathway is critical for cellular Caol (DAG) . IP3 int[10-4 M) ]. AnxA1 t U73343 .-10 M) increased secretion 2.0 ± 0.3 fold above basal (-7 M), but not U73143 (10-7 M). U73122, but not U73143, also significantly inhibited secretion stimulated by Cch (10-4 M), which was a positive control. These data show that AnxA1 stimulates PLC to increase [Ca2+]i and stimulate secretion.The effects of U73122 and U73143 on secretion were then determined. AnxA1 (10-5 M), an inhibitor of IP3 receptors on the ER (-9 M) added alone significantly increased [Ca2+]i by 160.6 ± 43.4 nM (2+]i increase was significantly decreased to 35.7 ± 9.6 nM after 30 minutes of incubation with 2APB. Cch (10-4 M) was used as a positive control and its stimulation of [Ca2+]i was significantly blocked by 2APB as well.To further explore the PLC pathway, goblet cells were incubated for 30 min with 2APB (10n the ER . AnxA1 (-10 M) increased secretion 2.0 ± 0.3 fold above basal (-4 M), the positive control.A similar effect of 2APB was found when secretion was examined. AnxA1 (10-7 M) before stimulation with AnxA1 (10-9 M) or a positive control Cch (10-4 M). [Ca2+]i was significantly increased when AnxA1 was added alone ). Cch-stimulated increase in [Ca2+]i was also significantly inhibited.Next, we investigated the effect of the PKC inhibitor RO317549. Goblet cells were incubated for 30 minutes with RO317549   significantly increased [Ca2+]i to a peak of 235.6 ± 50.0 nM (2+]i and was 97.6 ± 19.6 (-4 M) significantly increased [Ca2+]i by 203.5 ± 40.2 nM. KN 93, but not KN92, significantly decreased Cch-stimulated increase in [Ca2+]i.Free intracellular Ca2+/CaMK) . We havea2+/CaMK . To inve-10 M AnxA1-stimulated secretion from 1.6 ± 0.1 to 0.9 ± 0.1 fold above basal  for 30 minutes (-9 M) added alone significantly increased [Ca2+]i by 152.5 ± 37.2 nM. AnxA1-induced [Ca2+]i increase was not significantly blocked by UO126 at any concentration. Histamine (10-5 M), which is known to activate ERK 1/2 was used as a control . AnxA1(10-10 M) alone increased secretion by 2.0 ± 0.3 fold above basal  for 30 minutes before stimulating the cells with AnxA1 (10-9 M) , which is known to increase cAMP (2+]i was 548.1 ± 110.6 nM. H89 significantly blocked VIP-stimulated increase in [Ca2+]i and was 252.5 ± 39.6 nM.To determine if AnxA1 increases cAMP levels to activate protein kinase A (PKA) to increase i H89 did not significantly inhibit AnxA1-stimulated glycoconjugate secretion  alone significantly increased [Ca2+]i to a peak of 141.2 ± 50.0 nM (n=4). Neither preincubation with 1-but or t-but altered the effect of AnxA1 on the Ca2+ response. The increase in [Ca2+]i stimulated by the positive control carbachol (10-4 M) was significantly blocked by the active inhibitor 1-but, but not the inactive inhibitor t-but.Phospholipase D (PLD) is an important regulator of several critical aspects of cell physiology, and is involved in cell functions like endocytosis, exocytosis, cell migration and mitosis . We prevough PLD –12. To e-10 M) alone increased secretion 1.9 ± 0.3 fold above basal (-4 M) was significantly decreased by 1-but, but not t-but. Based on these findings we conclude that AnxA1 does not active the PLD pathway to stimulate an increase in [Ca2+]i but does activate PLD to stimulate secretion in cultured rat conjunctival goblet cells.When secretion was measured, AnxA1  for 30 min prior to stimulation with AnxA1 (-9 M) significantly increased peak [Ca2+]i by 530.0 ± 109.2 nM (n=4). Preincubation with AA at 10-6 M significantly altered the AnxA1-stimulated response that was 228.1 ± 60.7 nM. AA at 10-5 M did not significantly alter the AnxA1 response. The positive control Cch (10-4 M)-stimulated response was significantly decreased with AA 10-6 and 10-5 M from 542.6 ± 78.6 nM to 217.7 ± 65.4 nM and 257.1 ± 52.6 nM, respectively.Another phospholipase that is present and can be activated in goblet cells is the PLA2 pathway –12. To d-10 M) increased glycoconjugate secretion by 1.9 ± 0.3 fold above basal (-5 M) to 0.5 ± 0.4. Thus AnxA1 does not active the PLA2 pathway to stimulate an increase in [Ca2+]i but does activate PLA2 to stimulate secretion in cultured rat conjunctival goblet cells.AnxA1  in conjunctival goblet cells. In contrast, LXA4 increased [Ca2+]i at 10-8 M . The differential effect of AnxA1 and LXA4 concentration on [Ca2+]i is consistent with their binding to different areas on the ALX/FPR2 receptor.In the present study we found that AnxA1 was able to significantly increase [Cantration -12 M in 2+]i although AnxA1 does utilize these pathways to stimulate secretion. In conjunctival goblet cells AnxA1 did not activate ERK 1/2 pathway to increase [Ca2+]i, but rather more likely AnxA1 increased [Ca2+]i to activate ERK1/2. ERK1/2 activity can be Ca2+-dependent via activation of PLC by a G-protein-coupled receptor or can be independent of Ca2+ when activated by β-arrestin-dependent down regulation of G-protein-coupled receptors (2+]i (2+]i after activation of PLC. Similarly for AnxA1 activation of PLD, the rise in Ca2+ occurs before activation of PLD. An increase in Ca2+ and activation of PKC as could occur by PLC stimulation could activate PLD in the goblet cells as documented in a mast cell line (Furthermore, we found that AnxA1 was not dependent on either the ERK 1/2, PLA2, or the PLD pathway to increase i , 38, 50.ell line . The sigThe function of AnxA1 has been studied in a variety of disease models in order to explore this agonist’s potential as a novel anti-inflammatory therapeutic agent, including models of inflammatory bowel disease , rheumatIn conclusion, our findings demonstrate that the glucocorticoid effector protein AnxA1 has potential in prevention or as a new treatment for dry eye, allergic conjunctivitis and other forms of ocular surface inflammation through its effect on conjunctival goblet cells. AnxA1 works through activation of the ALX/FPR2 receptor to stimulate multiple signaling pathways, ultimately leading to the secretion of high molecular weight glycoconjugates including mucins into the ocular tear film. These mucins are a crucial component of a healthy tear film, and thus the regulation of mucin secretion from conjunctival goblet cells by AnxA1 is an important contributor to ocular surface health, and prevention of ocular surface disease and treatment of these diseases.This manuscript is dedicated to Robin R. Hodges who passed away on March 13, 2021. For over 30 years she managed the Dartt laboratory ensuring its excellence. She was respected and beloved by all.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 Schepens Eye Research Institute Animal Care and Use of Committee.AL, MO, JB, and RH performed experiments. AL, RH, TU, CS, and DD wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Norwegian Research Council to AL and by NIH R01 EY019470 to DD and RO1GM038765 to CS.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.


To study the relationship between DNA double-strand break (DSB) repair gene mutations and the risk of papillary thyroid microcarcinoma (PTMC).One hundred patients with PTMC or benign thyroid nodules (BTNs) at Henan Cancer Hospital were retrospectively analyzed. The DSB repair capacity of peripheral blood T lymphocytes in the two groups was assessed by flow cytometry. Data were compared using Student’s t-test to evaluate the relationship between DSB repair capacity and the risk of PTMC. Factors influencing DSB repair capacity were analyzed by multivariate logistic regression analysis. The relationship between PTMC and DSB repair capacity was analyzed by univariate analysis. Targeted next-generation DNA sequencing was applied to screen and analyze DSB repair genes related to PTMC.P < 0.05). Multivariate logistic regression analysis of age, sex, obesity status, radiation and other factors showed that radiation exposure was positively correlated with reduced DSB repair capacity. Moreover, univariate analysis showed that a reduction in DSB repair capacity was a risk factor for PTMC.Targeted next-generation DNA sequencing was performed on the DSB repair genes discovered, and those that were mutated in association with PTMC were Rad50 and FANCA; Rad51 mutations were related to BTN.The DSB repair capacity was 31.30% in the PTMC group and 44.40% in the BTN group, with that of the former being significantly lower (Radiation exposure is positively associated with induced DSB repair gene mutations, which may cause a reduced capacity for DSB repair and eventually lead to PTMC.The online version contains supplementary material available at 10.1186/s12935-021-02032-5. The incidence of thyroid cancer has increased in recent years, and it is currently the most common endocrine malignancy. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, accounting for approximately 80–90% . PapillaThe gene repair process is initiated to address DNA damage, and radiation is the most common exogenous factor causing such damage, which can destroy the integrity of the genome. At present, there are at least five DNA damage repair pathways known, namely, base excision repair, nucleotide excision repair, mismatch repair, DSB repair, and direct repair, among which DSB repair is very important . RadiatiBenefiting from advances in molecular biology and precision medicine, the spectroscopy probe designed by Agsalda Garcia  emits la2. The subjects were not related. After informed consent was obtained, a questionnaire survey was conducted among eligible subjects, and blood samples were collected.  ≥ 24 and 52 with BMI < 24. The pathological findings were PTMC and BTN in 50 patients each. Of the patients, 9 had a smoking history, whereas 91 did not. Regarding inclusion, all 100 patients with thyroid disease met the following criteria: (1) no history of any other cancer; (2) age 18 years and older; (3) no history of blood transfusion; and (4) no history of immunosuppressive therapy. Radiation exposure was defined as having received more than 3 X-ray examinations before the age of 12 years. Obesity was defined as BMI ≥ 24 kg/m7 cells) were isolated from 18 ml of whole blood by gradient centrifugation; the remainder was used for targeted next-generation DNA sequencing. All samples were processed within 8 h and frozen in liquid nitrogen for biomarker determination in batches.In this project, 20 ml of complete blood from each subject was collected into two 10 ml anticoagulant tubes with heparin. Lymphocytes (approximately 1 × 106 cells were divided into 8 parts) was put into 8 culture tubes of 12 mm × 50 mm: Four of them were DSB injury group (2 etoposide treated for 2 h and 2 drug-free treated tubes as control group);Four of them were DSB repair group  once, and then the culture medium without drug was used for 4 h, and two tubes without drug were used for control). Lymphocytes were treated in vitro with an experimental dose (20 μm) of etoposide. The cells were collected, fixed with 1% paraformaldehyde, washed with PBS once, and placed in 70% ethanol at − 20 °C for DSB detection.To detect DSB damage induced by etoposide, T lymphocytes were treated with phytohemagglutinin (PHA). Frozen lymphocytes were thawed rapidly and cultured at 37 °C for 48 h. Each cultured cell  fluorescent antibody was added and mixed. After 2 h at room temperature, the cells were centrifuged at 5000 BPM for 5 min; the supernatant was removed, and 1 ml BSA was added. The cells were mixed well and centrifuged at 5000 BPM for 5 min; the supernatant was removed, 200 µl RNA inhibitor was added, and 200 μl PBS buffer was added for flow cytometry to count fluorescence-positive and -negative cells.FastPure Blood DNA Isolation Mini Kit V2 (NanjingVazyme Company) was used. DSB repair genes such as Rad51, BRCA1, BRIP1, Rad50, FANCA, FANCD2, FANCI, TSC2, MSH3, MSH6, PRSS1, BARD1, and SDHA were sequenced based on second-generation probe capture methods. The sequencing data of the case group and control group were analyzed, and DSB repair genes related to thyroid cancer were screened and verified.2-test. The difference in etoposide-induced DSB repair capacity level between the PTMC and BTN groups was analyzed by the t-test. DSB repair capacity = 100 × {1 − [percentage of double-strand breaks after 4 h of drug treatment − percentage of double-strand breaks after 4 h of no drug treatment]/[percentage of double-strand breaks after 2 h of drug treatment − percentage of double-strand breaks after 2 h of no drug treatment/[percentage of double-strand breaks after 2 h of drug treatment − percentage of double-strand breaks after 2 h of no drug treatment] [P < 0.05.SPSS 22.0 software was used for statistical analysis. Differences in demographic variables  between the two groups were analyzed by the χeatment] . Binary 2-test (P > 0.05)  Table . DSB repP = 0.002) , though there was no significant correlation between age, sex, obesity history and DSB repair capacity (P > 0.05)  of the BTN control group. Logistic regression was used to analyze age, sex, obesity, radiation and other factors. It was found that radiation exposure was positively correlated with reduced DSB repair capacity .Further logistic regression analysis showed that the decrease of DSB repair capacity were positively correlated with the occurrence of PTMC  and nonhomologous end joining (NHEJ). According to the situation, HR and NHEJ can compete or cooperate to repair DSBs in eukaryotic cells. Defects in both pathways are closely related to disease, including immune deficiency and cancer susceptibility , 15. In P < 0.05), indicating that the observed decrease in DSB repair capacity was related to the increased risk of PTMC. Although the scale of this study was relatively small, the data showed that the PTMC patients had relatively low DSB repair capacity compared with the control group.The mechanism of many commonly used clinical antitumor drugs, including alkylating agents, DNA-intercalating agents, topoisomerase inhibitors, and antimetabolites, involves different forms of direct or indirect DNA damage. For example, etoposide acts as an inhibitor of topoisomerase II (Topo II), a homodimer that regulates the topological state of DNA by forming ATP-dependent DSBs. As Topo II is an enzyme necessary for cell survival, it has always been an important target of antitumor drugs , 17. RecIt is generally recognized that radiation is the most common exogenous factor causing DNA damage and inducing a variety of types of damage, including DSBs, single-strand breaks, base damage, purine- or pyrimidine-deficient sites, glycosyl damage and DNA protein cross-linking, among which DSBs are potentially fatal . DSBs caAdjadj  divided Rad50 is an important repair factor characterized by ATPase motifs at the N- and C-terminal regions, and the sequence between the two regions forms two helical domains adopting the shape of a long curl. Rad50 binds with Mre11 and NBS1 to form the complex Mre11-Rad50-NBS1 (MRN), which recognizes signals and initiates DSB repair . Xu 28]28] analyThe Fanconi anemia (FA) pathway is a complex network of functionally diverse proteins that is known for its role in mediating interstrand crosslink (ICL) repair . PalovcaIn KRAS mutation-driven colorectal cancer, Rad51 has been reported to be transcribed by the KRAS downstream target MYC and to participate in the irradiation-induced DNA damage response and repair . In the Overall, we believe that the reason why patients with reduced DSB repair capacity are more prone to thyroid cancer may be that DNA repair genes maintain the integrity and stability of the DNA genome by reversing DNA damage. Nevertheless, risk factors such as radiation can lead to genetic variation in DNA repair genes, which will impact the capacity for DNA repair and thus affect the risk of cancer.Because the cases in this study involved patient and control groups that comprised BTN patients, there was some genetic selection bias. For further study, a healthy control group will be included in our follow-up study. We found that reduced DSB repair capacity is associated with an increased risk of PTMC. However, we cannot exclude the influence of other potential confounding factors , which were not controlled in this study. Due to the small sample size, the relationship between DSB repair capacity or related repair gene mutations and the risk of PTMC needs to be confirmed by large-sample studies. In addition, key genes were not explored in this study. In future studies, we will investigate the function of Rad50 in the occurrence and progression of PTMC.In summary, this study focused on the role of DSB repair gene mutations in thyroid carcinogenesis and explored risk factors that reduce DSB repair capacity. The results showed that patients with low DSB repair capacity have a significantly increased risk of PTMC. In addition, history of radiation exposure is an important factor for a decrease in DSB repair capacity, which may be related to mutation of DNA damage repair genes as a result of radiation. Overall, we found that low DSB repair capacity may be associated with thyroid cancer. We identified Rad50 and FANCA as the most commonly mutated DNA repair genes. Future studies should verify the functions of these genes experimentally and identify other relevant mutated genes. Further studies should also search for simpler and more effective biomarkers for thyroid cancer risk assessment and provide a new scheme for the clinical treatment of thyroid cancer. An elevated level of clinical vigilance should be implemented for the high-risk population with low DSB repair capacity, as early detection through careful physical examination, B-ultrasound screening and close follow-up can improve prognosis (Additional files Additional file 1. Data of Clinicopathological characteristics of Benign Thyroid Nodules.Additional file 2. Data of Clinicopathological characteristics of Papillary Thyroid Microcarcinoma.


Mitochondria are central to many aspects of cellular metabolism providing energy, lipids, reactive oxygen species, signaling molecules for cellular quality control, and actively shaping intracellular Ca2+ signaling, modulating the intensity and duration of the signal itself. Abnormalities in the ability of mitochondria to take up and subsequently release Ca2+ could lead to changes in the metabolism of the organelle, and of the cell as a whole, that eventually result in cell death. We sought to investigate the role of mitochondria and Ca2+ signaling in a model of Familial Alzheimer’s disease and found early alterations in mitochondria physiology under stressful condition, namely, reduced maximal respiration, decreased ability to sustain membrane potential, and a slower return to basal matrix Ca2+ levels after a mild excitotoxic stimulus. Treatment with an inhibitor of the permeability transition pore attenuated some of these mitochondrial disfunctions and may represent a promising tool to ameliorate mitochondria and cellular functioning in AD and prevent or slow down cell loss in the disease.In Alzheimer’s disease (AD), the molecular mechanisms involved in the neurodegeneration are still incompletely defined, though this aspect is crucial for a better understanding of the malady and for devising effective therapies. Mitochondrial dysfunctions and altered Ca Biological hallmarks of the disease are extracellular deposition of amyloid plaques, composed mainly by fibrils of amyloid-ß (Aß) peptide, intracellular neurofibrillary tangles (NFTs) consisting of aggregated, hyperphosphorylated tau protein, and extensive neurodegeneration  due to a complex series of events: (i) Activation of AMPA receptors with depolarization and Ca2+ influx through voltage gated channels; (ii) opening of ionotropic NMDA receptors; and (iii) activation of Group I mGlu receptors. To evaluate the variations of intracellular Ca2+ content in cultured neurons after the toxic stimulus, WT and B6.152H neurons, 9 div, were infected with AA-virus expressing the cytosol targeted Ca2+ probe jRGECO . Although a ratiometric dye would be easier to calibrate in terms of [Ca2+], we chose jRGECO for these experiments for its fluorescence intensity and suitability to detect the Ca2+ changes due to neuronal action potentials [2+] changes with a non ratiometric probe can, however, be obtained by the measurements of the ΔF/F0 values. Being non ratiometric, in addition, means the probe is potentially affected by the pitfalls or artefacts due to movements of the focal plane or of the cells during the acquisitions, so extra care was taken to ensure stability of the focal plane, and movements of the cells were controlled for during offline analysis. The cells were challenged with the addition of 2 µM glutamate to mimic a sustained excitatory signal that elicited a large cytosolic Ca2+ increase. The rate of fluorescence increases, and the peak amplitude reached were comparable between the two genotypes, with no significant difference, indicating analogous endowment of cellular receptors and channels. After the rapid Ca2+ rise, its concentration progressively decreased to a lower plateau level; we then measured the jrGECO ΔF/F0 ratio 20 min post glutamate addition to investigate whether the cells were equally able to extrude excess Ca2+ from the cytosol and return toward the basal level. In the time frame of the experiment, we observed that both cell types reached a comparable cytosolic [Ca2+]. As discussed above, this probe does not allow the quantification of the absolute values of cytosolic [Ca2+] in the neurons, but the calculation of the ΔF/F0 permits one to compare the variation in [Ca2+] produced by the stimulus in the two cell types. Potassium chloride (KCl) 30 mM was added at minute 25, towards the end of acquisition, to further depolarize the plasma membrane, promote the opening of Voltage-Operated Channels (VOCs), and evaluate the residual capacity of the cells to accumulate Ca2+.Glutamate is considered the principal excitatory neurotransmitter in the brain , importae jRGECO  under thtentials . Informa2+ content, helping in finely shaping Ca2+ signals  changes within the mitochondrial matrix brought about by the glutamate challenge. For these experiments, WT and B6.152H neurons were infected with AAV expressing the mitochondrial Ca2+ probe 4mtD3cpv [2+], independently of the amount of probe expression level  could be due to the incapacity of the respiratory chain to work at a sufficient rate when challenged with a sustained ion entry. However, it could also be caused by the opening of the permeability transition pore. We checked whether the PTP was involved in the depolarization caused by the glutamate excitotoxicity by treating B6.152H neurons with a low concentration (1.5 μM) of the inhibitor Alisporivir (formerly known as Debio 025), which inhibits PTP opening via cyclophilin D binding, but, differently from its parent compound, cyclosporin A, does not bind to calcineurin  could be detrimental for mitochondria  within mitochondria is higher in B6.152H cells than in controls, despite no significant average difference in cytosolic [Ca2+] and a larger depolarization of the organelles in the transgenic cells. It needs to be stressed that these results are the average of several tens of cells, while it has been impossible to monitor Ca2+ levels  and mitochondrial membrane potential in the same cells. Accordingly, in cells with dramatic drops in mitochondrial membrane potential, the matrix [Ca2+] will not decrease to the basal level, but it will simply equilibrate with the cytoplasmic [Ca2+] i.e., it will be close to that in cells with normal mitochondrial membrane potential. On the contrary, in cells with only partial depolarization, the efficacy of Ca2+ efflux from the mitochondria via the Na+/Ca2+ exchanger  would be reduced [2+ would be higher compared to controls The average [Ca2+] in the mitochondrial matrix will thus depend on the percentage of B6.152H cells with the membrane potential identical, lower, or even completely depolarized compared to the controls. Most relevant, however, preventive incubation of the cells with a micromolar concentration of an inhibitor of the PTP delayed, and in some cases completely avoided, the decrease in the mitochondrial membrane potential suggesting that the PTP might be involved, and inferring that in the AD model, it could be more sensitive to stimuli promoting its opening. We judged the potentially protective effect of Alisporivir very interesting as it could be developed into a therapeutic tool for AD, and in vivo experiments on the FAD mouse model are presently being started by our group.A physiological increase in cytosolic Camembrane ,69, but eostasis . Moreoveeostasis . Upon th reduced , and the