Fanconi’s anemia (FA) is an inherited bone marrow failure syndrome that carries a higher risk of transformation to acute myeloid leukemia (AML) when compared with general population. AML is the initial presentation in approximately one third of patients.A 17 year old male presented to the emergency room with history of high grade fever since two weeks. Examination revealed pallor, short stature and thumb polydactyly. There was no visceromegaly or lymphadenopathy. Complete blood count showed haemoglobin 3.4 gm/dl, MCV 100 fl and MCH 36 pg, white blood cell count 55.9 × 10 E9/L and platelet count 8 × 10E9/L. Peripheral blood smear revealed 26% blast cells. Bone marrow was hypercellular exhibiting infiltration with 21% blast cells. Auer rods were seen in few blast cells. These findings were consistent with acute myelomonocytic leukemia. These blasts cells expressed CD33, CD13, HLA-DR, CD117, CD34 antigens and cytoplasmic myeloperoxidase on immunophenotyping. Bone marrow cytogenetics revealed 46, XY, t (8:21)  [11] / 46, XY, add (2) (q37), t  [4] / 46, XY [5]. Molecular studies showed positivity of FLT 3 D835 variant and negativity of NPM 1 and FLT3 ITD  mutation. Peripheral blood analysis for chromosomal breakage exhibited tri-radial and complex figures. He received induction chemotherapy with cytarabine and daunorubicin (3 + 7). Day 14 marrow revealed clearance of blast cells.The recognition of specific cytogenetic abnormalities present in FA known to predispose to AML is crucial for early haematopoietic stem cell transplant (HSCT) before transformation to leukemia. Fanconi anemia (FA) is an autosomal recessive disease which is the most frequent cause of inherited bone marrow failure syndromes. These patients usually present in the first and second decade of life  with bonDue to the increase in chromosomal instability, P53 activation and cell death; there is an increased possibility of clonal evolution in FA patients . The higThe leukemia in FA differs from that of general population. In one retrospective study , it was We report a case of young male who presented with AML and based on his physical abnormalities, peripheral blood was analyzed for chromosomal breaks. He had rare cytogenetic abnormalities not seen in AML transformed from FA.A 17 year old male presented to the emergency with a history of high grade fever since two weeks. Examination revealed short stature, tapering jaw Figure A. Bone mConsidering that the patient was high risk AML, option of subsequent allogeneic bone marrow transplant was discussed at consolidation therapy. Complete human leukocyte antigen (HLA) typing of the patient was performed and peripheral blood cytogenetics of his siblings was advised. He received induction chemotherapy with daunorubicin and cytarabine (3 + 7). During the hospital course, he developed febrile neutropenia for which he was empirically treated with antibiotics. Blood and urine cultures did not reveal growth of organisms. The neutropenia resolved on Day 21 and the patient became afebrile. Day 14 bone marrow revealed clearance of blast cells while minimal residual disease showed 0.03% blast cells. He was discharged and was advised follow up in outpatient clinic. However the patient was lost to follow up.We have described a case of FA AML (AML that is presumed to be transformed from FA) with rare cytogenetics and poor prognosis due to positivity of FLT3 D835 mutation. AML is 500 fold more likely to develop in FA patients than the general population  and apprThe most frequent chromosomal abnormalities in FA AML were gain of 1q, monosomy 7, and gain of 3q ,12 whileOur patient had none of the specific cytogenetic abnormalities known for FA AML except for t which is known to be more frequent in de novo AML and addition 2q, the significance of which is unknown. This indicates that AML should be considered as a transformation from FA even in the absence of known cytogenetic abnormalities for FA AML. This is important since FA cells are highly sensitive to chemotherapeutic agents leading to a poor outcome and the need for an early HSCT before transformation to leukemia occurs .The rate of absolute neutrophil count (ANC) recovery after chemotherapy is known to be associated with certain cytogenetic abnormalities , such asLiterature review has revealed that there have not been any prospective studies or case reports from our region and the prevalence of specific cytogenetic abnormalities in our population needs to be known. It remains unclear in this case whether the cytogenetic abnormalities in the leukemic cells were present in the bone marrow prior to the diagnosis of leukemia. It is possible that Fanconi Anemia predisposes to the evolution of leukemic clones. It also remains unclear whether routine cytogenetic testing of patients with FA would identify patients at increased risk of developing AML and who are in need of HSCT. Systematic long-term follow-up studies needs to be carried out in our population.This case emphasizes the importance of identifying patients with AML who have transformed from FA in the absence of specific cytogenetic abnormalities. The recognition of specific cytogenetic abnormalities present in FA known to predispose to AML is crucial for early HSCT before transformation to leukemia.Written informed consent was obtained from the patient’s father for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. This case has also been exempted for ethical approval by the ethical review committee of Aga Khan University and Hospital (2561-Pat-ERC-13).FA: Fanconi’s anemia; AML: Acute myeloid leukemia; FA AML: Acute myeloid leukemia transformed from Fanconi anemia; ANC: Absolute neutrophil count; ITD: Internal tandem domain; FISH: Fluorescent in situ hybridization; HLA: Human leukocyte antigen; MDS: Myelodysplastic syndrome; ALL: Acute lymphoblastic leukemia; HSCT: Haematopoietic stem cell transplant.The authors declare that they have no competing interests.SH collected the information and wrote the manuscript. SNA is the primary physician who reviewed and edited the manuscript. Both authors read and approved the final manuscript.SH is a fourth year resident in haematology department at the Aga Khan University and Hospital. SNA is an Associate professor and consultant haematologist at The Aga Khan University and Hospital.


Phytophthoracapsici is unable to synthesize its own ergosterol as the potential target of fungicide cinnamaldehyde (CA). In this study, CA exerted efficient inhibitory effects on both mycelial growth (EC50=0.75 mM) and zoospore germination (MIC=0.4 mM) of Pcapsici. . CA-induced immediate Ca2+ efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca2+ content determined by using Fluo-3 AM and the increase in extracellular Ca2+ concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca2+ influx with ruthenium red and verapamil led to a higher level of CA-induced Ca2+ efflux, suggesting the simultaneous occurrence of Ca2+ influx along with the Ca2+ efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca2+ gave rise to the impaired vitality of Pcapsici.  while the addition of exogenous Ca2+ could suppress the growth inhibitory effect of CA. These results suggested that Ca2+ efflux played an important role in CA-induced growth inhibition of Pcapsici. . The application of 3-phenyl-1-propanal, a CA analog without α,β- unsaturated bond, resulted in a marked Ca2+ influx in zoospores but did not show any growth inhibitory effects. In addition, exogenous cysteine, an antagonist against the Michael addition (the nucleophilic addition of a carbanion or another nucleophile) between CA and its targets, could attenuate CA-induced growth inhibition of Pcapsici.  by suppressing Ca2+ efflux. Our results suggest that CA inhibits the growth of Pcapsici.  by stimulating a transient Ca2+ efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.As a destructive fungus-like plant pathogen, the oomycete  Intracellular Ca2+ distribution is tightly controlled by active (ATP-dependent) membrane calcium pumps. Several types of perturbations in Ca2+ homeostasis will elicit cell death [2+ homeostasis was involved in CA-induced growth inhibition of Pcapsici. . Firstly, CA-induced inhibition of the growth of Pcapsici.  was accompanied with the increase in a rapid Ca2+ efflux in zoospores. Secondly, the addition of Ca2+ chelator EGTA resulted in the decreases in the intracellular Ca2+ level and the vitality of zoospores. Thirdly, the addition of exogenous Ca2+ not only compromised the decrease of intracellular Ca2+ level induced by CA but also significantly ameliorated CA-induced growth inhibition of Pcapsici. . Fourthly, treatment with PA  failed to induce Ca2+ efflux and growth inhibition of zoospores.CA is capable of stimulating a Call death ,30. Calcll death ,18, In t2+ level stored in cytoplasm or the pathway involving disturbance of calcium homeostasis triggers the fungal growth inhibition. Our results indicated Ca2+ influx induced by PA instead of CA did not lead to the growth inhibition of Pcapsici. . Thus it could be concluded that not all the disturbance to calcium homeostasis are fatal to the fungal growth, and the pathway should be especially concerned in the association between calcium homeostasis and antifungal action. Calcium efflux has been showed associated with broad-based fungicidal activity of amiodarone [Pcapsici.  by CA. So far, the plasma membrane is revealed containing two systems for Ca2+ efflux: Na/Ca2+ exchanger (NCX) and Ca2+-ATPase  [It is still unclear whether the state of Caiodarone . Calciump, PMCA) . Among tp, PMCA) ,34.2+/H+ Exchanger (CAX) exporting Ca2+ of the cytosol to maintain optimal ionic concentrations in fungi cell [Pcapsici.  (http://genome.jgi-psf.org/Phyca11/Phyca11.home.html), there were 16 potential Ca2+- pumps (Ca2+-ATPases), while none of CAX homologue could be found in the present genomic data of Pcapsici. . PMCA has been showed associated with inhibition of NFκB nuclear translocation and to promote cell death by regulating calcium signaling [2+ efflux via Michael additions with PMCAs of Pcapsici. , finally leading to its growth inhibition. Further research is needed to construct a platform for the independent expression of these Ca2+-ATPases and to determine the effects of CA on these ATPases.However, there is a Cangi cell . Based oignaling . Taken t


Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements  are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation.In 2+/HCO3- movement) and duodenum . We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel , 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels .A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels  and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters.We propose that the HCOGallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.Our  The prerequisite for shell mineralisation is the supply of large amounts of Ca2+ and CO32- in a limited extracellular milieu by trans-cellular transport, requiring the presence of ion channels, ion pumps and ion exchangers. In Gallus gallus, eggshell formation takes place daily in the hen uterus and is one of the most rapid mineralisation processes  is present. This channel is significantly overexpressed in the uterus compared with the magnum, where Ca2+ transfer is low. Its uterine expression is similar to that of the duodenum where Ca2+ absorption is also large. Cellular Ca2+ influx might use a similar Ca2+ channel, TRPV6, at the intestinal and uterine level but their localisation is hypothesized to differ according to the site of Ca2+ influx, being located in the basal membrane in the uterus but in the apical membrane in the intestine. The uterine expression of TRPV6 is not however modified according to whether calcification takes place. The presence of other Ca2+ channels cannot be ruled out as additional putative candidates. A recent transcriptomic study in our laboratory comparing uterine gene expression in hens with or without shell calcification revealed the presence of high expression of TRPC1, TRPP, TRPM7, TRPML1 and ORAI 1 .Caf uterus ,9 and innhydrase ,31, and nhydrase , made ithibitors -10 and thibitors . Trans-e protein  and acti[2+ pump . The higthe cell ,34. Our 2+ buffer is crucial to keep the free cytosolic Ca2+ concentration below toxic levels. Following Ca2+ entry into the uterine glandular cell, several systems could contribute to intracellular transport of Ca2+, while maintaining the low but essential free Ca2+ concentration for survival of the cell. In certain tissues, calbindin proteins, 9 kDa and 28 kDa in mammals [2+ binding capacity. Direct correlation has been demonstrated between their mucosal concentration and the efficiency of Ca2+ transfer in intestine and uterus under numerous experimental conditions [2+ and serve as a Ca2+ buffer needed for cell protection against Ca2+ stress and accompanying apoptotic cellular degradation that is induced by a high intracellular Ca2+ concentration [2+ transport from the basal membrane of the glandular cells to the apical membrane where Ca2+ is extruded into the uterine fluid.An intracellular Ca mammals  or 28 kD mammals ,35, are nditions ,28,30. Intration ,36,37. Intration ,14,28. T2+ concentration relies on the endoplasmic reticulum which contributes to Ca2+ homeostasis through its capacity for Ca2+ uptake and storage [2+ ATPases  play an active role in Ca2+ uptake by this organelle (reaching 10 to 100 mM free Ca2+), while maintaining the cytoplasmic concentration at low concentrations of 10-4 mM free Ca2+. Amongst the three isoenzymes  were overexpressed in the uterus compared to the magnum but were not modified when comparing the presence or absence of calcification. The higher expression of ITPR1 and ITPR 2 in the uterus compared to the duodenum supports our hypothesis concerning their contribution to the regulation of intra-cellular Ca2+. The ryanodine receptors which are involved in muscle excitation-contraction coupling in mammalian tissues [2+ release from the endoplasmic reticulum. RYR1 expression was revealed in the uterus, but there was no difference between the uterus, magnum or duodenum, suggesting a weak involvement in endoplasmic reticulum Ca2+ release. In conclusion, these observations of high expression of genes encoding ATP2A pumps and ITPR Ca2+ channels involved in Ca2+ uptake and release in endoplasmic reticulum suggest the involvement of this organelle in intracellular Ca2+ buffering in uterine glandular cells.The inositol 1, 4, 5-trisphosphate receptors (ITPR) are intracellular Caeticulum ,42 and a tissues  are alte2+ trans-epithelial transport is output from the glandular cells, which occurs against a concentration gradient. Ca2+ secretion towards the uterine fluid occurs via an active process, involving the Ca2+ ATPase [2+) [2+ secretion at the uterine level. In contrast, ATP2B1 and ATP2B4 were underexpressed in the uterus compared to duodenum and for ATP2B4 in presence of shell formation. In mammals, it is ATP2B1 which plays a more important role in intestinal Ca2+ absorption [2+-transporting ATPase 4 (ATP2B4) in the apical membrane of uterine epithelial cells but did not explore the presence of ATP2B2 and its differential expression during calcification. In human osteoblasts, the isoforms 1 and 2 take part in the Ca2+ supply necessary for bone mineralisation whereas the isoform 4 is not detected [The last step of uterine Ca+ ATPase ,32,43. Tase [2+) . Four isase [2+) . Only thsorption ,45. In osorption  localizedetected .+ transfer by Na+/K+ ATPase inhibitors considerably reduced Ca2+ secretion into the uterine lumen [2+ secretion and Na+ re-absorption. The uterine absorption of Na+ is revealed by the decreased Na+ concentrations in the uterine fluid observed between the early stage of shell calcification and the end of calcification  were overexpressed in the uterus compared to the magnum, whereas their expression did not change in the presence or absence of eggshell mineralisation  and the uterine fluid  and glandular cells at the basal membrane level will prevent Ca2+ uptake in the cells by exchange with Na+. Both Na+/Ca2+ exchangers (SLC8A1 and 3) are therefore predicted to be present only in the apical membrane of the uterine glandular cells. The co-expression of the SLC8A1 and 3 genes and of ATP2BX is observed in numerous Ca2+ transporting epithelia [2+ flux has been questioned. Na+/Ca2+ exchangers have a weak affinity for Ca2+, but strong Ca2+ conductance. On the other hand, the Ca2+ ATP2BX pumps have a strong affinity for Ca2+, but a weaker conductance [2+ transport is mainly assured by the Na+/Ca2+ exchangers. In the hen uterus, the inhibition of the Na+/K+ ATPase led to a 60% decrease in Ca2+ transport in vitro or during uterine perfusion [+/Ca2+ exchangers in the avian uterus.It was observed thirty years ago that the inhibition of Nane lumen ,17, showal level . Our stugradient ,47. Simipithelia -51 but tductance . These derfusion ,17. This2+ transport is summarized in the model described in FigureThe information on uterine Ca+ is absorbed from the uterine fluid into the blood plasma. This absorption resulting from the predominance of apical to basolateral flux relative to basolateral to apical flux, is partly due to the presence of the Na+/Ca2+ exchangers (SLC8A1 and 3), but a complementary system has been demonstrated by using epithelial Na+ channel blockers [. Amiloride-sensitive Na+ channels are essential in various epithelia [+ channel are overexpressed in the uterus compared to the magnum and to the duodenum  was overexpressed during shell calcification in contrast to the α and β subunits  suggesting its predominant involvement in the uterus.During eggshell calcification, Nablockers . Amiloripithelia . Three s+ glandular cell output towards plasma is active and occurs against a large electrochemical gradient , is present in the uterus whereas the α2 and α3 subunits  are absent. In this study, the α1 subunit (ATP1A1), but also the β1 subunit of Na+/K+ ATPase (ATP1B1), were overexpressed in the uterus compared to the magnum. We also confirmed the overexpression of α1 subunit of Na+/K+ ATPase during the phase of calcification in contrast to the β1subunit of Na+/K+ ATPase, in agreement with Lavelin et al. [In the basolateral membrane, the Naent Table. This isplacenta ,53. Its placenta -10,17. In uterus  showed tn et al. .+ from plasma into the uterine glandular cells at the basal membrane via Na+/HCO3- co-transporters  is discussed in the section addressing HCO3- transfer.The possibility of an uptake of Na+ channels provide the driving force for electrogenic transport processes across membranes and are involved in cell volume regulation or in secretory and reabsorptive processes. K+ channels are crucial for maintenance of body homeostasis and form the largest group of ion channels in mammals as more than one hundred thirty genes have been identified in human [+ channel candidates in chicken uterus by selecting 4 K+ channels overexpressed in uterus compared to their expression in magnum, as revealed in our hen transcriptomic study [+ concentrations in uterine fluid between early (8 hours) and late stages of calcification  but no experimental data has explored this contribution through a putative paracellular pathway. K+ uptake at the basolateral membrane, from the blood plasma towards the uterine glandular cells, should result from activities of the Na+/K+ ATPases . By analogy with other intestinal, kidney, pancreatic, placenta, mammary glands or blood cells, we expect that K+ channels present in the glandular cell will recycle K+ to allow functioning of the active Na+/K+ ATPases. We tested 4 genes: three coding K+ channels  and one K+ large conductance Ca2+ activated channel (KCNMA1), which could be involved in K+ cell output  participate in K+ output in a large range of tissues and epithelia and are regulated by Ca2+ cellular levels [+ channels were higher in the uterus compared with the magnum. Moreover, the expression of KCNJ2, 15 and KCNMA1 (KCNM subunit α) was stimulated during calcification  which penetrates the uterine glandular cells by simple diffusion through the plasma membrane [2 to HCO3-, which is secreted into the uterine fluid. In the mammalian gastrointestinal tract, including pancreas, the cellular and membrane bound CAs are key enzymes allowing secretion or reabsorption of large amount of acid across the mucosa or protect epithelial cells from acid injury by secreting bicarbonate [3- from CO2 at a higher rate than the stomach or distal small intestine. The CO2 originates from intestinal lumen at the duodenal level but is provided from blood plasma via the respiratory system to the uterine tissue [2 to produce HCO3- [3- uterine production. A major role for CA2 is supported by the overexpression of this CA gene in the presence of eggshell mineralisation, in contrast to CA4 and 7  have been located in apical membrane and Na+/HCO3- co-transporters (SLC26AX) at the basolateral membrane [3- secretion and Cl- transport [3-/Cl- exchangers. The HCO3- flow through the uterine apical membrane is an electrogenic process which is facilitated by output of intracellular Cl-, via an exchanger of the SLC26 electrogenic family [3-/Cl- exchanger (SLC26A9) in the uterus as shown in other epithelial cells [3- secretion, in particular to neutralise gastric acidity [3- for eggshell calcification. Na+/HCO3- co-transporter genes  are likely to have a minor role in HCO3- supply to the uterine glandular cells. The cystic fibrosis transmembrane conductance regulator (CFTR) contributes to fluid secretion from epithelial cells of the lung, pancreas and intestine, as shown in pathological situations associated with impaired fluid production, Cl- and HCO3- secretion due to defective CFTR [3- secretion is unlikely because of the unfavourable gradient or it is possibly indirect through regulation of HCO3- transporters [- channel is discussed in the following section on Cl-. Studies using specific inhibitors and measuring Cl- and HCO3- flows are needed to quantify the contribution of HCO3-/Cl- exchangers in HCO3- uterine secretion.The HCOpancreas  and in dpancreas  which semembrane ,59. In transport  which suc family ,57. Our  acidity ,61. Our , 7, 10; ,62) are membrane . The ion studies  showed tive CFTR ,64 or inive CFTR . Its consporters . Its rol3- production in the glandular epithelial cells, its secretion into uterine fluid and the co-precipitation of CO32- with Ca2+ leads to a progressive acidification of the uterine fluid and of glandular cells [+ are produced for each CaCO3 formed. This metabolic acidosis is partially compensated by hyperventilation by the hen and by an increased renal H+ excretion [HCOar cells ,5. In faxcretion .2+ -transporting ATPases  of the apical membrane actively extrude Ca2+, as previously mentioned. However several lines of evidence have established that these pumps contribute to H+ re-absorption coupled to Ca2+ secretion [2+ secretion by uterine glandular cells during eggshell formation and therefore in H+ re-absorption from the uterine fluid through the apical membrane. Alternatively, the Na+/H+ exchangers have been shown to contribute to H+ output in the pancreatic duct which also secretes large amount of HCO3-. In a recent transcriptomic study of the uterus , we detected expression of various Na+/H+ exchangers , supporting this possibility.The plasma membrane Caecretion ,67. The + ATPase pump (VAT) is expressed in the bird uterus during calcification. In mammals, this VAT complex is made up of at least 14 subunits and allows transfer of H+ by hydrolysis of ATP [3 biominerals and shown to export H+ during mineralisation [+ across the basolateral membrane of pancreatic duct epithelium [3- using mechanisms quite similar to uterine glandular cells. Our study reveals overexpression of the VAT subunit B in the uterus, which transfers large amounts of H+ compared with the magnum, where limited amounts of H+ are transferred. The VAT is likely to participate in H+ export from cytoplasm of the uterine cells to the blood plasma across the plasma membrane. The role of CLCN5 in H+ transfer is discussed in the ensuing Cl- section.In this study, RT-PCR shows that the V Hs of ATP ,69. Thiss of ATP . VAT is lisation ,72. Thisithelium  which is- concentrations decrease from 71 to 45 mM in the uterine fluid , 6 to 10 h after ovulation possibly through a paracellular pathway [+ and Cl- net fluxes are inversed. The net flux of Cl- is inhibited by acetazolamide, demonstrating the relationship between Cl- transport and HCO3- secretion derived from CAs activity [3-/Cl- exchangers of the SLC4 or SLC26 family [3-/Cl- exchangers, we observed no expression of SLC4A8 and there is no evidence of any expression of SLC4A1, 2 or 3 in avian uterine transcriptomic study [3- section. This exchanger is predicted to be located in the apical membrane of the uterine glandular cells and to contribute to Cl- cell uptake during eggshell calcification as suggested in hens subjected to acetazolamide inhibitors [The Clactivity ,9,31, an6 family ,62,73. Aic study . The rolhibitors ,59.- channel), is relatively ubiquitous in epithelial cells and other cellular types [- trans-epithelial transfer [- concentrations are favourable to a Cl- passive entry in uterine glandular cells. In parallel, another Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) could also contribute to Cl- entry in the cell as observed in numerous tissues [- entry in the cell.The CLCN2 channel, a family member of the CLCN  and 4 K+-Cl- cotransporters  are putative candidate, as expression of these genes is revealed in the chicken uterus transcriptome . In addition, furosemide, a blocker of Na+-K+-2Cl- cotransporters, has been shown to decrease egg shell thickness [On the other hand, Clby CLCN5 . The CLCxchanger  has beengulation -84. In thickness .2+ and HCO3- necessary for shell mineralisation , 28 kDa calbindin , endoplasmic Ca2+ pumps type 2, 3 , and inositol trisphosphate receptors type 1, 2, 3 . Ca2+ is then extruded from the glandular cells by the membrane’s Ca2+ pumps  and Ca2+/Na+ exchangers . The endoplasmic Ca2+ pumps, inositol trisphosphate receptors, and 28 kDa calbindin contribute to maintain a low intracellular free Ca2+ concentration essential for cell survival.(1) Ca+ transport involves three Na+ channels , Na+/Ca2+ exchangers SLC8A1 and 3 (uptake in the cell) and the Na+/K+ ATPase .(2) Na+ uptake entry into the cell results from the Na+/K+ ATPase; the K+ channels  contribute to its output release at the apical membrane.(3) K3- is mainly produced from CO2 by CA2 and to a lesser extent by CA4, and is also provided at a low level from plasma by the Na+/HCO3+ co-transporters . HCO3- is exported from the cell through the HCO3-/Cl- exchanger SLC26A9.(4) HCO3- synthesis in the cell and co-precipitation of HCO3- with Ca2+ in the uterine fluid produces two H+ which are transferred to plasma via the membrane Ca2+ pumps ATP2B1, 2 in the apical membrane and the VAT pump at the basolateral level.(5) HCO- ions in the uterine fluid enter the cell by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels , and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+-K+-2Cl- and K+-Cl- cotransporters (SLC12Ax).(6) Cl2+ and HCO3- or in maintaining cellular homeostasis . The model qualitatively describes putative mechanisms and cellular localisation of the candidates. These hypotheses relying on expression of the genes and on analogies with other tissues that transfer large amount of ions, need to be confirmed using immunochemistry for their cell localisation or by specific inhibition, to establish their relative contribution and understand their interaction and regulation. This avian model where huge amounts of Ca2+ and HCO3- are exported daily following a precise spatial and temporal sequence should contribute to understanding the mechanism and regulation of ionic precursors of CaCO3 and provide insight for other species secreting a CaCO3 biomineral such as coral, molluscs, foraminifera or sea urchins.This model proposes a large but not exhaustive list of ionic transfer proteins involved in the supply of CaThe experiment was conducted at the Unité Expérimentale Pôle d'Expérimentation Avicole de Tours  according to the legislation on research involving animal subjects set by the European Community Council Directive of November 24, 1986 (86/609/EEC) and under the supervision of an authorized scientist . Forty week old laying hens (ISA brown strain) were caged individually and subjected to a light/dark cycle of 14 hour light and 10 hour darkness (14 L:10D). The hens were fed a layer of mash as recommended by the Institut National de la Recherche Agronomique (INRA). Each cage was equipped with a device for automatic recording of oviposition time.gastrocnemius) were harvested in 8 hens while the egg was in the uterus during the active phase of calcification (16–18 hour post-ovulation). Additionally, uterine tissues were collected from 8 birds injected with 50 μg of F2-α prostaglandin during 4 consecutive days to expel the egg before mineralisation had begun (6 to 8 hours post ovulation). All tissue samples were quickly frozen in liquid nitrogen and stored at −80°C until RNA extraction.Tissue samples . The quality of the primers was tested by virtual PCR for dimerization and specificity using Amplify 3X software [The list of ion transporters was established using recent transcriptomic data and ers Table, were desoftware .Gallus gallus RefSeq nucleic data bank.Total RNA was extracted from frozen tissue samples using a commercial kit  and simultaneously treated with DNase  according to the manufacturer’s procedure. RNA concentrations were measured at 260 nm using a NanodropND 1000 . The integrity of RNA was evaluated on a 2% agarose gel and with an Agilent 2100 Bioanalyser . Only RNA samples with a 28S/18S ratio > 1.3 were considered for RT-PCR and qRT-PCR experiments. Total RNA samples (5 μg) were subjected to reverse-transcription using RNase H-MMLV reverse transcriptase  and random hexamers . PCR was performed using primers . A one-way analysis of variance was performed to detect differences  in gene expression amongst different conditions.Alternatively, cDNA sequences were amplified in real time using the qPCR Master mix plus for SYBR® Green I assay  with the ABI PRISM 7000 Sequence Detection System . To account for variations in mRNA extraction and reverse transcription reaction between samples, mRNA levels were normalized either to ribosomal 18S rRNA levels for each sample in the first series of comparison  or to TBP (TATA box binding protein) for each samples in the second series of comparison . The expression levels of 18S rRNA were measured using TaqMan Universal PCR Master Mix and developed Taq-Man assay for human 18S rRNA  as previously validated . The PCRThe authors declare that they have no competing interests.VJ, JG contributed to the strategy, the experimental design, and planning of the study. VJ carried out the experiments and analyses, interpreted data and wrote the first draft of the paper. JG is the supervisor of VJ (Ph.D. student). AB contributed to the interpretation of data and to the writing of the paper. YN conceived the research program focused on identification of egg proteins. He was involved in the strategy, the experimental design, data interpretation and was fully involved in the writing of the paper. All authors have read and approved the final manuscript.RT-PCR of the candidate genes potentially involved in ion transfer in four secreting tissues and in muscle.Click here for file


Although complementation with βSG gene was inconsequential, knock-in of miRNA669a (missing in βSG−/− cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of βSG−/− cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca2+-handling properties of βSG−/− cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca2+-spark properties and RyR isoform expression, distinguished βSG−/− cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in βSG−/− cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by βSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from β-sarcoglycan-null mice (βSG The timing rate .2+ release events (Ca2+ sparks) were recorded at ×63 magnification in line-scan mode (sampling rate 0.8 kHz). Images were analysed by SparkMaster (ImageJ) software o). Furthermore, the properties of Ca2+ sparks occurring in electrically quiescent cells (spontaneous releases) were studied in the three cell types. To focus on SR-dependent Ca2+ release, all measurements were carried out in the absence of extracellular Ca2+ (Ca2+-free solution).Sarcoplasmic reticulum (SR) function as a Ca2+ responses in βSG−/− and C2C12 myotubes  and infrequent in CMs , where FDHM never exceed 60 msec. In βSG−/− myotubes the same release site generated both sparks and embers; this was at variance with C2C12 myotubes, where each site generated a single class of events (i.e. spark or ember). Parameters means for all release events are compared between the three cell types in −/− myotubes, all the parameters sharply distinguished CMs events from those of βSG−/− and C2C12 myotubes, which were similar to each other. In the average, CMs events had larger amplitude, faster kinetics and smaller width as compared with those of the other cell types. Further characterization of elementary Ca2+ release events between the three cell types may be provided by comparison of distributions of spark image parameters, provided in the Supplement  were subjected to the same differentiation protocol and studied at the same times as non-transfected ones. R-βSG−/− cMabs failed to form myotubes retaining a morphology similar to the wild-type cMabs. Ca2+ responses were evoked by ATP, but these cells were insensitive to either caffeine or nicotine  dependency of voltage-triggered contraction on extracellular Ca2+; (2) presence of cholinergically induced contraction and Ca2+ responses; (3) membrane current and potential responses to cholinergic stimulation compatible with a robust expression of receptor-operated channels of the nicotinic type (blocked by d-tubocurarine); (4) ICaL kinetics and activation voltage dependency; (5) features of elementary Ca2+ release events, both in terms of average parameters and of their distribution patterns ; (6) expression of Cav 1.1 and RyR1 transcripts, with virtual absence of Cav 1.2 and RyR2 ones.The findings of this study indicate that many functional properties assimilate βSG−/− myotubes displayed many functional properties distinctive of skeletal muscle and, under homogeneous experimental conditions, shared them with myotubes formed by a line of skeletal muscle precursors (C2C12 cells). This observation is consistent with a robust commitment of the βSG−/− mutant to the skeletal phenotype, as revealed by our previous molecular characterization [−/− cMabs after rescue (by miRNA699a transduction); thus, it can be considered specific of the mutant genotype. At variance with previous studies [−/− cMabs with miRNA669a, aimed to rescue the effects of βSG deletion, prevented skeletal differentiation but, at least in vitro, it failed to reinstate cardiac one [etc.) inconsistency of cardiac differentiation of WT cMabs between the present and previous studies [−/− and WT cMabs were studied after the same number of passages (see methods), cell ageing cannot be responsible for the discrepancy.βSGrsors C2C cells. T studies , we werersors C2C cells. Trsors C2C cells. T studies  has no o−/− and C2C12 myotubes were found to differ under several, more subtle, aspects. As compared with C2C12 myotubes, βSG−/− ones had (1) larger variability in repolarization, presence of AP subpopulations with a discernible plateau phase; (2) higher frequency of elementary Ca2+ release events, which included more than one amplitude class and occurred as sparks and embers within the same release site; (3) absence of RyR3 expression; (4) slower depletion of Ca2+ store by repeated excitations.Even if sharing features typical of skeletal muscle, βSGto, a repolarizing current appearing late during myocyte maturation [−/− myotubes might point to persistence of a cardiac trait in an otherwise skeletal phenotype. As ICaL is the main current supporting the AP plateau, biphasic repolarization might depend on partial persistence of its cardiac isoform. However, in βSG−/− myotubes ICaL kinetics and the Ca2+ channel isoform expression were typically ‘skeletal’. Moreover, Cav1.2 channel expression, obvious in CMs, could not be detected in βSG−/−  seemingly exhausting the releasable Ca2+ pool (2+ pool not accessible to VICR operation [−/− myotubes, which do not express RyR3, may suggest the presence in these cells of a RyR1 population uncoupled from membrane voltage sensing.The absence of RyR3 expression and slower depletion upon repeated excitations might be causally related. Indeed RyR3 channels are supposed to amplify VICR by adding a CICR component , 28. The events. . Thus, af events ; interesa2+ pool b. This operation . The occ−/− cMabs function might suggest persistence of such a commitment.The present findings lead to conclude that deletion of the βSG encoding gene, which includes the miRNA699a/q sequences, removes an inhibitory control on cMabs differentiation towards a skeletal functional phenotype. Rescue of deletion effects by transduction of miRNA699a suggests that the latter is involved in the inhibitory control. These findings provide a functional counterpart to the previously reported role of miRNA699a in preventing skeletal-type gene expression in cMabs . Althoug


Aims. To investigate whether changes of meat consumption can affect body composition and laboratory parameters in healthy, normal weight, young women without the aim to reduce body weight. Research Design and Methods. Women volunteered to eat low-fat meat in addition to their habitual diet (M) or to exclude meat products from their diet (NOM). After 4 weeks M and NOM were crossed over between subjects. Changes in nutrient intake, morphometrics and plasma parameters were compared during M and NOM. Results. Daily protein intake (means ± SD) was 2.25 ± 0.35 (25.2% of energy) and 1.15 ± 0.26 g/kg (14.0% of energy) during M and NOM, respectively. Fat-free body mass (FFM) increased during M  and decreased during NOM . Body fat mass was unchanged. Concentrations of total cholesterol (−7%), LDL-cholesterol (−8%), and glucose (−4%) deceased significantly after M. Fasting glutamine concentrations were decreased by M and increased by NOM. Conclusions. Additional meat intake can increase FFM without adverse effects on blood lipid concentrations. Long-term studies are required. Urinary excretion of 3-methylhistidine could represent a biomarker for meat protein consumption. High-protein diets are suggested to increase satiety, to benefit fat oxidation, to reduce energy efficiency during overfeeding, and to contribute to a better conservation of fat-free body mass (FFM) which can support diet therapies of biomedical problems such as obesity or loss of FFM –4. For tFourteen female subjects were recruited among students of the Institute of Nutritional Sciences at the University of Potsdam. Inclusion criteria were good health, age between 20 and 30 years old, a habitual protein intake of about 1 g/kg/d, and no excessive intake of semiluxury food. Exclusion criteria were infectious, metabolic, and gastrointestinal diseases, food intolerances, eating disorders, pregnancy and lactation, extreme forms of dieting, and regular usage of drugs. After having received verbal and written information about the study, all subjects gave written consent. The study protocol was approved by the Ethics Committee of the University of Potsdam in accordance with the Helsinki declaration .The study had a randomized, crossover design, where 4 weeks of additional intake of exclusively low-fat pork fillet (M) and an exclusion of meat and meat products (NOM) was examined, respectively. Baseline measurements were executed. After this seven of the subjects started the first dietary intervention period M (group A). Seven other subjects started with NOM (group B). After four weeks the interventions M and NOM were crossed over between subjects. During period M, participants were asked to eat the 200 g of lean pork fillet per day in addition to their usual diet. Pork fillet was bought by participants from local supermarkets and butcher's shops and costs were compensated. The origin of pork fillet was recorded. Participants were allowed to distribute the additional meat intake during M over the whole day. However, it was recommended not to be eaten together with the usual meals. This was done to ensure that the meat was eaten additionally to the habitual subjects' diet and that it was not used only for a replacement of their usual portions. Individual portions were reported in a protocol. During NOM participants were asked to abstain from consumption of all meat and meat products but were allowed to eat eggs and dairy products.http://www.dife.de/). The coding of nutrient intake  was carried out on the basis of the German Food Code and Nutrient Data Base BLS II.3 . Dietary intake information was assessed by a semiquantitative and self-administered 4-d food record from Sunday to Wednesday prior to the study and during the fourth week of each intervention period . The rec2 within 30 min of blood collection, and stored at −80°C before analysis. 24-hour collections of urine were made at baseline and within week 3 of each intervention period. Nitrogen excretion was measured in these samples.Subjects visited the institute between 0800 and 1000 h after an overnight fast at baseline and after 4 wks of each intervention period for anthropometric measurements and for sample collection. Blood samples were drawn into Li-heparinized tubes  which were placed on ice within 10 min. Plasma was separated by centrifugation , frozen in liquid N2 (m2)] was calculated. Body fat mass and fat-free body mass (FFM) were measured by air displacement plethysmography  according to the manufacturer's recommendations. Anthropometric measures were obtained by trained personnel while subjects wore no shoes and only light underwear. Body weight was assessed on an empty bladder using a digital scale to the nearest 100 g . Body height was measured with a flexible anthropometer to the nearest 0.1 cm . Body mass index (BMI) homocystine (98%)  as an internal standard [Concentrations of cholesterol, triacylglycerol, glucose, and insulin were measured using colorimetric or enzymatic standard methods. Nitrogen content of urine was determined using an elemental analyzer (intra-assay CV < 3%) . Amino acid and urea concentrations in plasma and urine were analyzed by ion-exchange chromatography as reported previously (intra-assay CV < 5%) . The detstandard .n = 14). The means after M and after NOM were tested for significant differences by dependent (paired samples) t-test. In addition, changes were calculated between subject-specific values after either M or NOM in relation to values before M or NOM, namely, baseline or end of first intervention period. The significance of the resulting effect of M or NOM calculated as change of individual values was computed using the 2-tailed one sample t-test with zero as hypothetical mean value. The relationships between variables were estimated by Pearson correlations . Regression analysis was used to evaluate the relationship between dietary data and measured data. Standardized regression coefficients were tested by t-test for deviation from the null hypothesis. The significance threshold was set at P = .05. We used SPSS for Windows  or WinSTAT  for statistical evaluations.All data were tested for normal distribution with the Shapiro-Wilk test for normality. One value of FFM which was obviously an outlier possibly due to false data input was excluded from calculations. Descriptive analysis includes means ± SD. Mean values are presented for baseline, M, and NOM . Furthermore, an endogenous 3MH excretion of ∼90 μmol/d was computed relating the values of daily 3MH excretion of all participants against the animal protein intake at baseline, during M and during NOM based on 4-d food records . Dietary macronutrient and energy intake were reported in The total fat consumption was significantly higher during M as compared to NOM. However, in terms of energy percentage, fat contribution was not significantly different. The total carbohydrate consumption was not significantly different between M and NOM. However, carbohydrates provided about 10% less energy throughout M as compared to NOM. The mean total energy intake was significantly higher during M by about 290 kcal/d compared to NOM.2, P = .027) and decreased during NOM . However, the significant increase in body weight by about 0.7 kg during M (P = .02) and the decrease in body weight by about 0.8 kg during NOM (P = .7 × 10−4) was based exclusively on changes of FFM. In addition, there was a positive correlation of changes in total protein intake with changes in BMI  and FFM (The BMI was significantly higher after M compared to NOM . The BMI and FFM .P < .05) after M by 7%, 8%, and 4%, respectively  and total cholesterol  (data not shown). The fasting mean THC concentrations in plasma were also not significantly different between M and NOM and were ~10 μmol/L . However, there revealed a negative correlation between changes in total protein intake during both M and NOM and individual changes in plasma glutamine concentrations  or changes in glutamine to cystine ratios  (data not shown) which was suggested to be a marker of skeletal muscle catabolic state [μmol/L) and positive (+38 ± 60 μmol/L) individual changes of fasting glutamine concentrations after M and NOM, respectively. Nevertheless, the mean fasting concentrations of indispensable amino acids lysine and valine were significantly higher after M compared to NOM . Although, the biological or metabolic significance of this effect cannot be explained sufficiently it has been reported that catabolic conditions may result in lower plasma concentrations of glutamine or glutamine/cystine ratios [There is an ongoing discussion about pathogenic roles of amino acids, for example, in the development of insulin resistance –9, 25. We ratios , 32. Finally, we detected similar THC as well as methionine concentrations 4 weeks after exposure to either M or NOM. Methionine is known as the sole precursor of THC. However, the calculated methionine intake has been considerably lower during NOM as compared to M . The resLimitations to our research include the fact that meat is not only a source of dietary protein. Among other nutrients it was proposed that iron derived from red meat may increase iron stores and consequently, could initiate oxidative damage and inflammation. This was suggested to be responsible for long-term adverse effects resulting in coronary heart disease and type 2 diabetes. However, a replacement of dietary carbohydrate with protein from red meat by an addition of lean red meat of approximately 215 g/d was not shown to elevate several parameters responsible for oxidative stress and inflammation after an exposure of 8 weeks . In addiIn summary, a 4 weeks exposure to 200 g lean meat added to the habitual diet of healthy young woman resulting in high-protein intakes seems not to produce adverse effects on body composition, concentrations of blood lipids, insulin, glucose, and THC compared with an exclusion of all meat products. The additional meat consumption may be effective in enhancing FFM with no change in fat mass. The urinary 3MH excretion may serve as a reliably biomarker for the consumption of meat protein provided that the validity was assessed in further studies. Further research is needed to assess the specific mechanisms that explain the possible benefits or risks of long-term high-protein (meat) consumption for nutrition-dependent diseases.


DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene,  Caenorhabditis elegans as a model system. Here we describe a new gene, dsb-1, that is required for double-strand break formation in C. elegans. Through analysis of the encoded DSB-1 protein we illuminate an important regulatory pathway that promotes crossover recombination events on all chromosome pairs to ensure successful meiosis.For most eukaryotes, recombination between homologous chromosomes during meiosis is an essential aspect of sexual reproduction. Meiotic recombination is initiated by programmed double-strand breaks in DNA, which have the potential to induce mutations if not efficiently repaired. To better understand the mechanisms that govern the initiation of recombination and regulate the formation of double-strand breaks, we use the nematode  Formation of crossovers between homologous chromosomes is essential for successful execution of the meiotic program in most sexually reproducing organisms. In addition to shuffling genetic information between parental chromosomes, crossovers, together with cohesion between sister chromatids, create physical links between homologous chromosomes that enable their segregation to daughter cells during the first meiotic division Saccharomyces cerevisiae, the activities of cell cycle-regulated kinases involved in DNA replication ensure that that DSBs occur only after DNA synthesis is complete S. cerevisiae, and Drosophila melanogasterMeiotic recombination is initiated by programmed DNA double-strand breaks (DSBs), a subset of which is repaired to form crossovers between homologous chromosomes  have known orthologs in other phyla; and even these two proteins are absent in several species, including Caenorhabditis elegans, D. melanogaster, and Neurospora crassaMeiotic DSBs are catalyzed by the widely conserved, topoisomerase-related enzyme Spo11 C. elegans has emerged as a valuable model system for molecular analysis of meiosis. As in other eukaryotes, SPO-11 catalyzes the formation of meiotic DSBs S. cerevisiaeC. elegans, as in other species, meiosis-specific chromosome architecture contributes to DSB proficiency. In particular, in the absence of HTP-3, an integral component of chromosome axes, DSBs are abolished or sharply reduced C. elegansC. elegans meiosis are unusual among model organisms, including the fact that synapsis between homologous chromosomes is independent of recombination C. elegans will likely reveal both conserved aspects of meiosis and how regulatory circuits are remodeled during evolution.The nematode dsb-1 (double-strand break factor 1), that is required for meiotic DSB formation in C. elegans. dsb-1 mutants lack meiotic DSBs, and show meiotic defects similar to spo-11 mutants. DSB-1 localizes to meiotic chromosomes coincident with the time of DSB formation, in a manner dependent on the CHK-2 kinase. We also find that a variety of mutations that disrupt crossover formation on one or more chromosomes extend the chromosomal localization of DSB-1, suggesting that the DSB-permissive state may be prolonged. Based on these observations, we infer the existence of a regulatory circuit in which meiotic nuclei monitor the recombination status of each chromosome pair and act through DSB-1 to maintain a DSB-permissive state until all chromosome pairs have attained crossover-competent recombination intermediates.Here we identify a novel gene, C. elegans, mutations that impair meiotic chromosome segregation result in embryonic lethality and a high incidence of males (XO) among the surviving progeny dsb-1(we11) mutant was isolated in a genetic screen for maternal-effect embryonic lethality, and was found to produce a high fraction of males among its few surviving self-progeny. A targeted deletion allele of the affected gene, dsb-1(tm5034), was generated independently (see below), and results in defects identical to dsb-1(we11) based on all assays described here. Whereas self-fertilizing wild-type hermaphrodites produce nearly 100% viable progeny and 0.2% males , , , Table 1broods), , Table 1dsb-1 mutants are quantitatively similar to several previously characterized mutants that fail to make any crossovers during meiotic prophase, such as spo-11  mutants, except that mutations in dsb-1 were associated with reduced brood size (dsb-1(we11) showed linkage to the middle of Chromosome IV, close to the spo-11 locus, complementation tests revealed that we11 is not an allele of spo-11. Quantitative RT-PCR also indicated that spo-11 mRNA levels were unaffected in dsb-1(we11) mutants (The defects observed in ood size . Althoug mutants .dsb-1(we11) animals identified several mutations in annotated coding sequences, including a nonsense mutation in the previously uncharacterized gene F08G5.1 (F08G5.1 expression via transgene-mediated cosuppression we11 mutation affects this gene. we11 introduces a premature stop (tac = >taa) after lysine 96 ((tm5034) removes 290 bp from predicted exons 3 and 4 and the intervening intron (dsb-1(tm5034) mutants is indistinguishable from dsb-1(we11)  . Both arCaenorhabditis, including other nematode genera. Interestingly, the genomes of C. elegans and several other Caenorhabditids each contain 2 predicted paralogs. In an accompanying paper, Rosu et al. show that dsb-1 paralog F26H11.6/dsb-2 is also involved in meiotic DSB formation in C. elegansCaenorhabditis, members of this protein family are not well conserved (The DSB-1 protein has no apparent homologs outside of the genus onserved .C. elegans ORFs DSB-1 lacks identifiable domains that might give clues about its function in DSB formation. One notable feature is its high serine content: 60 of 385 amino acids (16%) are serine residues, compared to an average serine content of 8% encoded by all E. coli. Immunofluorescence staining revealed that DSB-1 is absent from somatic nuclei, and specifically localizes to chromosomes during early meiotic prophase  mutant spo-11(ok79) mutants, which lack functional protein C. eleganshtp-3 mutants, which lack an essential axial element component that is important for DSB formation We tested whether DSB-1 localization depends on other factors required for DSB formation. DSB-1 localized to meiotic chromosomes in the catalytically dead C. elegans, including DSB formation and homolog pairing chk-2(me64) mutants . However, the reduction in DSB-1 protein levels in chk-2 mutants does not appear to fully account for the sharply diminished chromosomal localization of DSB-1. These data indicate that DSB-1 localization to chromosomes is largely dependent on the CHK-2 kinase, and suggest that DSB-1 may act downstream of CHK-2 to promote DSBs.The CHK-2 kinase is essential for several key events during early meiotic prophase in  mutants . Althougrec-8 and rad-54 mutants, in which DSB-1 staining disappeared by late pachytene, but RAD-51 staining persisted into diplotene cosa-1(me13)and zhp-3(jf61)com-1(t1626)rad-51(Ig8701)rad-54(tm1268)syp-1(me17)syp-2(ok307)We quantified the extension of DSB-1 localization by comparing the length of the zone of DSB-1-positive nuclei to the total length of the region spanned by the transition zone through late pachytene nuclei, just before oocyte nuclei begin to form a single row near the bend region of the gonad, which coincides with diplotene . We desiad-54tm12syp-1(mespo-11(ok79 or me44)mre-11(ok179)rad-50(ok197)Significantly, mutants that lack meiotic DSBs, including htp-1 and htp-3 mutants Also of note, in spo-11 mutants. Young adult hermaphrodites were irradiated, then fixed and stained 8 hours later. As controls, we included mutants (mre-11 and msh-5) in which crossover defects are not rescued by exogenous DSBs spo-11(me44) animals to 56%, compared to 70% for unirradiated controls , msh-5, syp-2, him-8, and zim-2 (see below) were combined with ced-4(n1162), which abrogates germline apoptosis Many mutations that result in extension of the DSB-1 zone also cause elevated oocyte apoptosis, which can be triggered in response to persistent DNA damage or asynapsis him-8(tm611) and zim-2(tm574) specifically disrupt homolog pairing and thus crossover formation on chromosomes X and V, respectively him-5(ok1896) does not impair pairing or synapsis, but abrogates DSBs on the X chromosome him-5 animals, in which the crossover defect can be rescued by exogenous DSBs him-8, suppressed the extension of DSB-1 localization  or by staining for synaptonemal complex components (in him-8 mutants). In both of these genotypes, we observed persistent DSB-1 staining on all chromosomes throughout the region of extended DSB-1 localization  mutants, although the fluorescence intensity was reduced relative to wild-type gonads . The nature of this biased distribution shows interesting variation among species Proteins with apparent homology to DSB-1 are restricted to the C. elegans distinguish it from other organisms in which DSB-promoting factors have been identified. In particular, DSBs and early recombination steps contribute directly to homolog pairing and synapsis in many species, while in C. elegans homolog pairing and synapsis occur independently of DSBs. Additionally, C. elegans lacks Dmc1, Hop2, and Mnd1, which are thought to function together as an essential meiotic recombination module in most eukaryotes C. elegans also lacks the DSB proteins Mei4 and Rec114, which are conserved between budding yeast and mice Several features of meiosis in C. elegans strains were cultured under standard conditions at 20°C. The wild-type strain was N2 Bristol. The nonsense dsb-1(we11) allele was generated by EMS mutagenesis. The dsb-1 deletion allele (tm5034) was generated by the Japanese National BioResource for the Nematode. Both dsb-1 alleles were extensively outcrossed to wild-type (5-6x), and additionally outcrossed in a directed three-point cross to dpy-20 unc-30 to eliminate most linked mutations. Additional mutants analyzed in this study were: spo-11, mre-11(ok179), rad-50(ok197), chk-2(me64), com-1(t1626), rad-51(Ig8701), rad-54(tm1268), msh-5(me23), cosa-1(me13), zhp-3(jf61), htp-3(tm3655), htp-1(gk174), syp-1(me17), syp-2(ok307), him-8(tm611), zim-2(tm574), dsb-2(me96), and ced-4(n1162). Strains used in this study were:All dsb-1(we11) IV/nT1[unc-?(n754) let-?] CA1104 dsb-1(tm5034) IV/nT1[unc-?(n754) let-?] CA1105 spo-11(me44)/mIs11 IVCA279 spo-11(ok79)/mIs11 IVCA276 mre-11(ok179) V/nT1[unc-?(n754) let-?] CA1109 rad-50(ok197) V/nT1[unc-?(n754) let-? qIs50] AV158 chk-2(me64) rol-9(sc148)/+ VCA1110 unc-32(e189) com-1(t1626)/qC1 dpy-19(e1259) glp-1(q399) IIIGE4132 rad-51(Ig8701)/mIs11 IVCA538 rad-54(tm1268) I/hT2[bli-4(e937) let-?(q782) qIs48] CA855 msh-5(me23) IV/nT1[unc-?(n754) let-?] AV115 cosa-1(me13)/qC1[dpy-19(e1259) glp-1(q339) qIs26] IIIAV424 zhp-3(jf61) I/hT2[bli-4(e937) let-?(q782) qIs48] CA685 htp-3(tm3655) I/hT2[bli-4(e937) let-?(q782) qIs48] CA821 htp-1(gk174) IV/nT1[unc-?(n754) let-?] AV393 syp-1(me17) V/nT1[unc-?(n754) let-? qIs50] AV307 syp-2(ok307) V/nT1[unc-?(n754) let-?] AV276 him-8(tm611) IVCA257 zim-2(tm574) IVCA258 dsb-2(me96) IIAV477 rol-1(e91) dsb-2(me96)/mnC1[dpy-10(e128) unc-52(e444)] IIAV539 rol-1(e91) dsb-2(me96)/mnC1[dpy-10(e128) unc-52(e444)] II; dsb-1(tm5034)/mIs11[let-?] IVCA1111 rol-1(e91) IICB91 ced-4(n1162) IIICA1087 ced-4(n1162) III; spo-11(ok79)/mIs11 IVCA1090 ced-4(n1162) III; msh-5(ne23) IV/nT1[unc-?(n754) let-?] CA1092 ced-4(n1162) III; syp-2(ok307) V/nT1[unc-?(n754) let-?] CA1091 ced-4(n1162) III; him-8(tm611) IVCA1088 ced-4(n1162) III; zim-2 (tm574) IVCA1989 unc-119(ed3) IIIDP38 L4 hermaphrodites were picked onto individual plates and transferred to new plates every 12 hours, for a total of 6–8 12-hour laying periods, until newly-laid fertilized eggs were no longer observed. Eggs were counted immediately after each 12-hour laying period. Surviving hermaphrodite and male progeny were counted 3 days later.E. coli using Ni beads under denaturing conditions. The protein was resolved on an SDS-PAGE gel and the excised DSB-1 band was used to immunize guinea pigs. Rabbit anti-HTP-3 antibodies were raised against a synthetic peptide (PTEPASPVESPVKEQPQKAPK) by Strategic Diagnostics Inc., SDIX. Additional antibodies used in this study were: guinea pig anti-HTP-3 Polyclonal antibodies against recombinant full-length DSB-1 protein were produced at Pocono Rabbit Farm & Laboratory. 6xHis-DSB-1 was purified from Immunofluorescence was performed as previously described For quantification of DAPI-staining bodies in oocytes, animals were dissected, fixed, and DAPI-stained as described above, omitting the steps involving immunostaining.FISH procedures have also been previously described in detail All images were acquired using a DeltaVision RT microscope (Applied Precision) equipped with a 100× 1.40 oil-immersion objective (Olympus) or (for whole gonad images) a 60× 1.40 oil-immersion objective (Olympus). Image deconvolution and projections were performed with the softWoRx software package (Applied Precision). Image scaling, false coloring, and composite image assembly were performed with Adobe Photoshop. All micrographs presented in the figures are maximum-intensity projections of 3D data stacks.Lysate from 50 young adult hermaphrodites, picked at 24 hours post L4, was used for each lane. Gel electrophoresis was performed using 4–12% Novex NuPage gels (Invitrogen). Proteins were transferred to PVDF membrane. Guinea pig DSB-1 antibodies and rabbit DSB-2 antibodies (see above) were used for immunoblotting, followed by detection with HRP-conjugated secondary antibodies and ECL Western Blotting Substrate (Pierce).Young adult worms were irradiated with approximately 10 Gy (1000 rad) from a Cs-137 source. For each experiment, unirradiated controls were treated identically to irradiated animals, other than exposure to radiation. For quantification of DAPI-staining bodies at diakinesis, hermaphrodites were irradiated 4–5 hours post L4 and dissected 18 hours post irradiation. To assess progeny survival, animals were irradiated 4–5 hours post L4, eggs laid 20–30 hours post irradiation were quantified, and surviving progeny were quantified 3 days later. For quantification of DSB-1 localization, animals were irradiated 16 hours post L4 and dissected 8 hours post irradiation. For RAD-51 immunofluorescence, animals were irradiated 24 hours post L4 and dissected 1 hour post irradiation.we11 animals were picked from an outcrossed, balanced strain. A genomic DNA library was prepared as described in the genomic DNA library protocol from Illumina. Libraries were sequenced using 76-bp single-end Illumina sequencing. MAQGene we11 mutant strain.1000 homozygous dsb-1 coding sequence and promoter was amplified by PCR using the following primers: 5′-CCGCTTCCGAATACCGCC-3′ and 5′-GGTGCCGCTGTGTAGAAGAAGC-3′. 100 ng/µl of dsb-1 PCR product was combined with 50 ng/µl of unc-119 rescuing plasmid pMM051 unc-119 animals. Rescued non-Unc F1 animals were picked to individual plates and assayed for embryonic lethality and male progeny. F2 animals were dissected, stained, and observed to quantify the number of DAPI-staining bodies in oocytes at diakinesis.A 2.1-kb region of genomic DNA including the spo-11 mRNA levels were compared by real-time PCR analysis with SYBR Green (Kapa Biosystems). act-1 and htp-3 mRNA levels were used as normalization controls. Primers used were as follows: spo-11 , act-1 , and htp-3 .12 young adult animals, 24 hours post L4, were used for each genotype. RNA was purified from animals and reverse transcribed into cDNA with the SuperScript kit from Invitrogen using poly-A primers. Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.Figure S3(TIF)Click here for additional data file.Figure S4(TIF)Click here for additional data file.Figure S5(TIF)Click here for additional data file.Figure S6(TIF)Click here for additional data file.


Background: PHLPP1 is a tumor suppressor phosphatase.Results: WDR48 in complex with USP12 controls cell survival signaling by deubiquitinating PHLPP1.Conclusion: WDR48 acts as a potential tumor suppressor by positively regulating PHLPP1 stability.Significance: WDR48 was identified as novel PHLPP1 regulator, which is critical in understanding its role as tumor suppressor in human cancers. PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival. The phophatidylinositol 3-kinase/Akt pathway is known to be hyperactivated in many human cancers . Many moin vivo. Regulation of PHLPP1 protein stability via ubiquitination was recently identified as one of the mechanisms to control PHLPP1 function in cells. β-TrCP-containing Skp·Cullin 1·F-box protein (SCF) complex (SCFβ-TrCP) was identified as an E3 ubiquitin ligase that ubiquitinates and degrades PHLPP1 in a phosphorylation-dependent manner  were PCR-amplified and cloned into S-protein/FLAG/streptavidin-binding protein (SFB)-triple-tagged destination vector using the Gateway cloning system (Invitrogen). Full-length WDR48 and USP12 were cloned to a Myc-tagged destination vector. HA-PHLPP1 and GST-tagged PHLPP1 vectors were generated by transferring their coding sequences into destination vectors using the Gateway system. USP12 catalytically inactive mutant (C48S) and WDR48 mutants were generated by PCR-based site-directed mutagenesis.Anti-PHLPP1 (Bethyl Laboratories), anti-USP12, anti-GST, anti-Myc, clone 9E10 , anti-pAkt (Ser-473), anti-Akt, anti-phospho-p70 S6K (Thr-389), anti-p70 S6K, anti-phospho-S6 (Ser-235/Ser-236), anti-S6 , anti-WDR48, anti-FLAG, anti-actin, anti-HA , and anti-ubiquitin (Millipore) antibodies were used in this study.m Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) containing 50 mm β-glycerophosphate, 10 mm NaF, 1 μg/ml of each pepstatin A, and aprotinin on ice for 30 min. After removal of cell debris by centrifugation, crude cell lysates were incubated with streptavidin-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. The bound proteins were washed three times with NETN and then eluted twice with 2 mg/ml biotin (Sigma) for 60 min at 4 °C. The eluates were incubated with S-protein-agarose beads (Novagen) for 1 h at 4 °C and then washed three times with NETN. The proteins bound to S-protein-agarose beads were eluted by boiling in SDS-loading dye and then resolved by SDS-PAGE. The identities of eluted proteins were revealed by mass spectrometry analysis performed by the Taplin Biological Mass Spectrometry Facility at Harvard.PHLPP1-associated proteins were isolated by using tandem affinity purification as described before . BrieflyHEK 293T, HeLa, and HCT116 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. Cells were transfected with various plasmids using polyethyleneimine transfection reagent. For immunoprecipitation assays, cells were lysed with NETN buffer. The whole cell lysates obtained by centrifugation were incubated with 2 μg of specified antibody bound to either protein A or protein G-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. The immunocomplexes were then washed with NETN buffer four times and applied to SDS-PAGE. Immunoblotting was performed following standard protocols.Bacterially expressed GST-PHLPP1 or control GST bound to glutathione-Sepharose beads (Amersham Biosciences) were incubated with 293T cell lysates for 1 h at 4 °C. The washed complexes were eluted by boiling in SDS sample buffer and separated by SDS-PAGE, and the interactions were analyzed by Western blotting.m) for 6 h. The whole cell extracts prepared by NETN lysis were subjected to immunoprecipitation of the substrate protein. The analysis of ubiquitination was performed by immunoblotting with ubiquitin antibody.Cells were transfected with various combinations of plasmids as indicated in The vector containing WDR48 shRNA (5′-AATAACATAGGAAACCTGCAC-3′) and USP12 shRNA (5′-AAACAGACGAAGTTCTAAAGG-3′) was transfected, and 48 h after transfection the cells were collected, and the efficiency of knockdown was checked by immunoblotting with specific antibodies.1 peak.HCT116 cells were transfected with various indicated vectors. Twenty-four hours after transfection the cells were washed with PBS and then treated with propidium iodide-hypotonic lysis buffer . After 30 min of incubation, the samples were analyzed by flow cytometry, and the percentage of apoptotic cells was calculated based on the sub-GHCT116 cells were transfected as required and then seeded into 100-mm or 60-mm dishes. The rate of cell proliferation was analyzed by counting the viable cells after staining them with trypan blue at the indicated times.A) in addition to several previously identified PHLPP1-associated proteins such as hScrib -tagged version of PHLPP1 (SFB-PHLPP1). Tandem affinity purification from the lysates of these cells using streptavidin-agarose beads and S-protein-agarose beads followed by mass spectrometry analysis was performed. We identified WDR48 as one of the major associated proteins in the PHLPP1 complex A in addis hScrib  and SUGTs hScrib . WDR48 . Interestingly, we observed that PHLPP1 specifically associated with WDR48 and USP12 but not other USPs . Although USP1 . In addition, bacterially expressed GST-PHLPP1 but not GST alone pulled down WDR48 . PHLPP1 has several domains that are critically important for its function. To identify the WDR48 interacting region of PHLPP1 we generated a series of N-terminal or C-terminal deletion mutants of PHLPP1 that lack different domains (A). Co-expression of these constructs along with full-length Myc-tagged WDR48 followed by immunoprecipitation suggests that WDR48 interacts with PHLPP1 within a region between its phosphatase domain and PDZ domain (residues 908–1202 amino acids) (B). However, to map the interacting region of PHLPP1 in WDR48 we also generated deletion mutants of WDR48, which contain or lack WD repeats (C). Co-expression of these constructs along with full-length PHLPP1 followed by immunoprecipitation suggested that the C-terminal region (devoid of WD repeats) is required for its binding to PHLPP1 (D).In addition to WDR48, we also observed that deubiquitinating enzyme USP12 in the list of PHLPP1-associated proteins. As PHLPP1 was already known to be ubiquitinated by β-TrCP , we hypoFig. 1ugh USP1  were rec domains A. Co-expo acids) B. Howeve repeats C. Co-expo PHLPP1 D.A). Similarly, USP12 knockdown also resulted in increased accumulation of PHLPP1 ubiquitinated species (B), suggesting that WDR48-associated USP12 might be required for deubiquitination of PHLPP1. The enzymatic activity of USP12 is required for PHLPP1 deubiquitination as PHLPP1 was significantly deubiquitinated by wild type but not the catalytically inactive USP12 (USP12 C48S) (C). However, PHLPP1 deubiquitination was enhanced by overexpression of full-length WDR48 but not the mutant (ΔWIR) that lacks the region of PHLPP1 interaction, indicating that PHLPP1 deubiquitination was dependent on its interaction with WDR48 (D).Because WDR48 is known to assemble and activate deubiquitinases to regulate its downstream substrates, we further tested whether the WDR48·USP12 complex controls PHLPP1 ubiquitination. Knockdown of WDR48 by using specific shRNA resulted in significant accumulation of polyubiquitinated PHLPP1 A. Simila species B, sugges12 C48S) C. Howeveth WDR48 D.E). In contrast, shRNA-mediated knockdown of WDR48 drastically reduced the protein half-life of PHLPP1 (F), clearly suggesting that the WDR48·USP12 complex stabilizes PHLPP1 by promoting its deubiquitination.Because polyubiquitination of PHLPP1 was known to regulate its protein stability, we further assessed whether the WDR48·USP12 complex-mediated deubiquitination of PHLPP1 alters its protein levels. In a cycloheximide chase experiment, we observed that PHLPP1 levels were stabilized to a greater extent in presence of WDR48 and USP12 compared with PHLPP1 alone E. In conf PHLPP1 F, clearlA). Similarly, shRNA-mediated knockdown of USP12 also resulted in increased activation of Akt (B) with no apparent changes in the total Akt levels. Surprisingly, no significant changes were observed in phosphorylation of p70 S6 kinase and S6 ribosomal proteins upon knockdown of WDR48 and USP12, possibly suggesting that the WDR48 complex may mediate its functions mainly through an Akt-dependent but p70 S6K- and S6-independent manner. The destabilization of PHLPP1 and activation of AKT upon depletion of WDR48 are dependent in proteosomal activity as these events could be restored with MG132 treatment (C). As knockdown of WDR48 and USP12 increased Akt activation, we next tested whether WDR48 and USP12 could potentiate the suppression of Akt signaling by PHLPP1. In fact, overexpression of WDR48 and USP12 together with PHLPP1 resulted in reduction of Akt phosphorylation, supporting the synergistic effect of the WDR48·USP12 complex along with PHLPP1 in suppressing Akt signaling (D). The synergistic effect of the WDR48·USP12 complex with PHLPP1 in suppressing Akt signaling is again dependent on the catalytic activity of the deubiquitinase as inactive mutant of USP12 shows no synergy in reducing Akt activation.Because PHLPP1 is a potent negative regulator of the PI3-kinase/Akt pathway, we hypothesized that WDR48 and USP12 might suppress Akt signaling by stabilizing PHLPP1. To test this hypothesis, we depleted WDR48 by using shRNA and analyzed the activation of Akt. Indeed, knockdown of WDR48 led to significant increase in the levels of phosphorylated Akt A. Similan of Akt B with noreatment C. As knoignaling D. The syE). Thus, these results suggest that WDR48 and USP12 negatively regulate Akt signaling and thereby induce apoptosis in conjunction with PHLPP1.It is well known that PHLPP1 being a tumor suppressor promotes apoptosis by negatively regulating Akt. Because the WDR48·USP12 complex stabilized PHLPP1 and acted in synergy with PHLPP1 in reducing Akt activation we hypothesized that expression of these proteins might also result in cellular apoptosis. In fact, overexpression of WDR48 and USP12 induced apoptosis similarly to PHLPP1. In agreement with their synergy with PHLPP1 function, simultaneous expression of WDR48 and USP12 along with PHLPP1 in cells led to significant increase in apoptosis compared with expression of PHLPP1 alone E. Thus, A) and analyzed their rate of proliferation. As shown in Fig. 5B, knockdown of WDR48 and USP12 by shRNA accelerated the rate of cell proliferation compared with control shRNA-transfected cells. In contrast, overexpression of WDR48 and USP12  suppressed cell proliferation similarly to PHLPP1 .PHLPP1 acts as a tumor suppressor by negatively regulating the Akt pathway. Thus, WDR48·USP12, being a deubiquitinase complex and a positive regulator of PHLPP1 stability, might function as a tumor suppressor complex as well. To test this possibility, we depleted WDR48 and USP12 by shRNA in HCT116 cells A and anaE and F), suggesting that inactivation of Akt is a crucial downstream event for WDR48-controlled cell proliferation.By using inhibitor studies we further tested whether WDR48-mediated suppression of cell proliferation is dependent on Akt inactivation. In fact, the augmented Akt activation and cell proliferation observed upon knockdown of WDR48 and USP12 were significantly reduced by treatment of cells with wortmannin , E and FA). Because the mutation is located in the C-terminal PHLPP1 interacting region of WDR48, we hypothesized that mutant inability to stabilize PHLPP1 might be due to the loss of their interaction. In fact, the WDR48 L580F mutant is severely defective in binding with the substrate PHLPP1 compared with wild type WDR48 (B). However, the mutant interaction with USP12 is unaffected (C). Consistent with the interaction data, unlike wild type WDR48 the L580F mutant is unable to effectively deubiquitinate PHLPP1 (D). In addition, L580F mutant is defective in suppressing Akt phosphorylation unlike wild type WDR48 (E). Further, the L580F mutant significantly failed to suppress the rate of tumor cell proliferation (F) compared with wild type WDR48. Together, these results suggest that WDR48 acts as a potential tumor suppressor by stabilizing PHLPP1, and mutations that disrupt its interaction with substrates may contribute to tumorigenesis.To further establish the role of WDR48 as a tumor suppressor and its dependence on PHLPP1 regulation, we screened for WDR48 mutations in human cancers that might be defective in regulating this pathway. Our search for somatic mutations in the COSMIC database revealed several missense and nonsense mutations in WDR48. Among these mutations, we identified that an L580F mutation of WDR48 found in colon adenocarcinoma is defective in stabilizing PHLPP1 A. Becauspe WDR48 B. Howeveaffected C. Consise PHLPP1 D. In addpe WDR48 E. Furtheferation F compareIn summary, we identified WDR48 and USP12 as two novel players in PHLPP1 regulation. We demonstrated that the WDR48·USP12 complex deubiquitinates and regulates PHLPP1 levels, which is essential for their possible tumor suppressor function. Because PHLPP1 is an important player in controlling cell survival and metabolism, it is critical to maintain the homeostasis of its protein levels and function in the cell. Any alterations in its levels and/or function may lead to diseases. For instance, loss or reduced PHLPP1 promotes proliferative pathways and is frequently associated with cancer, whereas gain of PHLPP1 results in termination of metabolic signaling pathways and neuronal cell survival pathways, which are often associated with insulin resistance in diabetes  and neurSo far, very limited mechanisms have been identified for regulation of PHLPP1 in cells. Whereas regulation of PHLPP1 at transcript level is completely unknown, its regulation at translational and post-translational levels is reported. At the translational level PHLPP1 is controlled by an mTOR-dependent pathway whereas at the post-translational level it is regulated by calpain-mediated proteolytic cleavage and β-TrCP-mediated proteasomal degradation. However, the enzymatic machinery is required to maintain proper PHLPP1 protein levels in cells by opposing β-TrCP mediated PHLPP1 degradation. USP1  and USP4Although WDR48 has been well established as an activatory subunit of different deubiquitinase complexes such as USP1, USP12, and USP46, its complete cellular functions and role in human malignancies have not been clearly demonstrated. In our study, in addition to identifying WDR48 as a physical partner of PHLPP1 in cells, functionally we also demonstrated its ability to act as a potential tumor suppressor. In fact, we identified a disease-associated WDR48 mutation in colon adenocarcinoma that is defective in binding and maintaining optimal PHLPP1 levels in cells. Other studies have previously shown that WDR48 is found in complex with different proteins such as PCNA, BRCA1, p53, FANCD2, and FANCI , 26, all