The bone mineral density correlated positive with the pooled ultimate load  for all constructs and negatively with angulation  throughout cyclic loading. Solid fracture‐specific and cannulated headless compression screws provide equal ultimate loads and stiffness for Jones fracture fixation. A low bone mineral density significantly impairs the construct stability and the ultimate load of both intramedullary screw constructs. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:911‐917, 2020Implant choice is a matter of concern in athletes and active patients who sustain a Jones fracture because they are prone to failure including non‐union, screw failure, and refracture. The aim of this study was to compare the biomechanical behavior of a Jones fracture‐specific screw (JFXS) with a cannulated headless compression screw (HCS) in a simulated partial weight‐bearing and ultimate load Jones fracture fixation model. Ten matched pairs of human anatomical specimens underwent Jones fracture creation and consecutive intramedullary stabilization with a solid JFXS or a cannulated HCS. The bone mineral density was assessed prior to testing. Cyclic plantar to dorsal loading was applied for 1000 cycles, followed by load to failure testing. Angulation was measured by an opto‐electronic motion capture system and mode of failure classification was determined by video analysis. Paired analysis showed no statistically significant difference between both screw constructs. Ultimate load reached 236.9 ± 107.8 N in the JFXS group compared with 210.8 ± 150.7 N in the HCS group ( Jones fractures or proximal fifth metatarsal fractures (Zone II and III) are common injuries in competitive and recreational athletes.In order to aim for clarification with regard to biomechanical aspects of screw types used in Jones fracture treatment, we compare a solid Jones fracture‐specific screw with a cannulated headless compression screw in a biomechanical Jones fracture fixation model by simulating initial post‐operative weight‐bearing and ultimate loading.2. Prior studies have reported an excellent repeatability for BMD evaluation using Lunar Prodigy densitometer.Ten matched pairs of fresh human foot specimens  were used for this biomechanical study. Donor age ranged from 64 to 92 years (mean 78.8 ± 8.7 years). The specimens were obtained from voluntary donors who consented to donate their body for research and teaching purposes to the Medical University of Vienna during lifetime. Institutional Review Board approval was granted prior to the conduction of the study (EK 2077/2013). The specimens were stored at –80°C and thawed at +4°C 48 h prior to testing to prevent tissue dehydration. Ahead of specimen preparation and biomechanical testing, dual‐energy X‐ray absorptiometry (DEXA) scans of the calcaneus were carried out to determine BMD. Areal BMD measurements were taken with Lunar Prodigy series X‐ray  and reported as g/cmThe fifth metatarsals were dissected and disarticulated from the feet Fig. A and B. The JFXS group received a solid partially threaded titanium screw with a small low‐profile head, especially designed for Jones fracture treatment  Fig. A. This sIn the HCS matched pairs, a countersinkable cannulated partially threaded titanium screw  was implanted Fig. B. This sThe small matched pair specimens (6/10) that received a 4.5 mm JFXS on one side received a 4.5 mm HCS screw on the other side accordingly. Vice versa we proceeded with larger specimens (4/10) and implanted 6.0 mm JFXS or 6.5 mm HCS screws.An experimental setup was designed to simulate post‐operative in vivo load conditions after surgical Jones fracture treatment. The fifth metatarsal specimens were potted with their proximal aspects in Wood's metal in 40 mm diameter custom built steel cups. The Jones fracture site was kept outside of the molding material. Wood's metal is a moldable bonding material for biomechanical testing and it was proven to be superior compared with polymethylmethacrylate (PMMA),The load was applied at the plantar aspect of the fifth metatarsal head. The specimens were loaded in compression from plantar to dorsal in a force‐controlled manner at 3 N/s up to 12 N. On the supposition that early post‐operative mobilization would involve a partial weight‐bearing regimen, 12 N has been chosen as the peak load. This load represents one‐half of the load to which the head of the fifth metatarsal is subjected during the normal push‐off phase of gait.After completion of cycle 1000, an ultimate load test was performed with the same force rate as described above. The failure of the construct was defined by exceeding 10° of interfragmentary angulation or until gross construct failure occurred . In constructs that failed before loading cycle 1000, the load applied at the time of failure was defined as the ultimate load.Modes of failure were classified after completion of the load to failure testing. Each specimen was liberated from Wood's metal fixation and inspected by two orthopedic surgeons (M.W. and R.S.). The mode of failure was determined upon agreement of both investigators after visual inspection of the specimens and video recordings. Standardized photographs were taken of each construct failure .Statistical analyses were performed using SPSS 20.0 for Windows , and the level of significance was defined as α < 0.05. Descriptive data were reported as means with standard deviation. All data showed a normal distribution in Kolmogorov–Smirnov test.t test was used to determine the significance of difference in stiffness (slope), displacement, angulation, and ultimate load between the experimental groups at each loading cycle.Paired samples t test.Intergroup differences between screw diameters were analyzed using independent Pearson product‐moment correlation coefficient was calculated in order to investigate the relationship between BMD and angulation, stiffness, and ultimate load.p ≤ 0.986; displacement: 0.131 ≤ p ≤ 0.635). The mean stiffness in the JFXS group and in the HCS group reached 40.6 ± 7.5 and 40.3 ± 13.5 N/mm at load cycle 1000, respectively (p = 0.928)  or the cannulated headless compression screw (HCS group) at any point during the cyclic loading (stiffness:0.324 ≤ 2) and it sustained a plantar metatarsal shaft fracture. According to preliminary failure criteria, we found a construct survival of 100% in the JFXS group and 90% in the HCS group. Matched pair analysis showed no statistically significant difference between the screw groups (0.238 ≤ p ≤ 0.600)  . The HCS diameters differed statistically significantly with a mean of 133.5 ± 59.1 N in 4.5 mm screws and 326.9 ± 180.0 N in 6.5 mm screws concerning the ultimate load (p = 0.037).When comparing screw diameters we found 202.1 ± 91.5 N load until failure in 4.5 mm and 289.3 ± 121.8 N in 6.0 mm solid JFXS constructs , followed by loosening of the screw head . The screw head cut out was defined as a sharp cut through the screw head in the proximal aspect of the fifth metatarsal. The screw was still rigidly embedded in the bone, but not at the original place of insertion. In contrast to a cut out of the screw head, we also found loosening of the screw head in some specimens. The screw head was loose in the proximal bone with rather a bony defect around the head.The most common mode of failure in HCS constructs was proximal screw head cut out , followed by screw head cut out . One JFXS construct showed a rotational instability at the proximal aspect of the fifth metatarsal. In this construct, a 4.5 mm screw was used and the specimen had a low BMD of 0.208 g/cm2  and 0.399 g/cm2  in the HCS group. There was no statistically significant difference between the groups (p = 0.839) and BMD did not correlate with age . There was a statistically significant difference in BMD between male and female specimens. The mean BMD in male specimens reached 0.446 g/cm2 compared with 0.315 g/cm2 in female specimens (p = 0.032). BMD showed a positive correlation with the pooled ultimate load  for all constructs. It correlated negatively with angulation , which was significant for every load cycle among all tested constructs . We also found a positive correlation of BMD with the stiffness during the initial phase of cyclic loading . The later loading cycles showed no significant correlation with the stiffness groups (0.121 ≤ p ≤ 0.287).The mean BMD in the JFXS group was 0.389 g/cmThe Jones fracture is a common sports injury, especially in professional athletes, and fast return to sports and competition is the primary aim of surgery.p = 0.429) though results were not statistically significant. In terms of stiffness (slope of the load–displacement curve) and angulation, we observed no difference in paired analysis between the JFXS and the HCS. Screw head cut out and screw head loosening comprised the most common modes of failure. This mode of failure was observed more frequently in HCS than in JFXS. We also observed higher initial angulation during cyclic loading in the HCS group in comparison with the JFXS group . One possible explanation for these observations could be that the engaging threads of the HCS head cut through the cancellous bone in the metaphysis of the fifth metatarsal and therefore the screws loosen easier during loading. Orr et al.We found higher ultimate loads during load to failure testing for the Jones fracture‐specific screw (236.9 ± 107.8) compared with the headless compression screw (210.8 ± 150.7 N)  is available in the diameters 4.5, 5.5, and 6.0 mm. The commonly obtainable and for multiple fracture fixations applicable cannulated Headless Compression Screw (HCS) is produced in diameters 4.5 and 6.5 mm. In order to compare equal screw sizes in this matched pair study, we used 4.5 mm JFXS or HCS in smaller specimens and compared a 6.0 mm JFXS to a 6.5 mm HCS fixation in larger specimens. We are aware of this mismatch. Nevertheless, this conflict reflects the surgeon's dilemma in the clinical routine, which is also limited by available implants and diameters.Data of the present Jones fracture fixation model shows that both screw constructs  provide equal ultimate loads and stiffness. In addition, low BMD seems to be related to a debilitated primary stability and diminished ultimate load in intramedullary Jones fracture fixation.M.W.: Designing the study, preparing the specimens, performing the experiments, analyzing the data, statistical analysis, figure design, drafting, revising, and approving the manuscript. E.B.: Performing the experiments, drafting, revising, and approving the manuscript. L.H.: Preparing the specimens, revising and approving the manuscript. M.F.K.: Statistical analysis, figure design, revising and approving the manuscript. G.B.: Preparing the specimens, performing the experiments, drafting and approving the manuscript. S.Z.: Radiological investigations, approving the manuscript. R.W.: Revising and approving the manuscript. R.S.: Designing the study, surgical procedure, revising and approving the manuscript. Each author represents that he has read and approved the final manuscript.


Zizania latifolia extract (ETZL), which protects against UVB-induced skin-aging. However, the suppressive mechanism of tricin on allergic responses remains unknown. The present study, therefore, aimed to determine the mechanisms of tricin and ETZL on mast cell degranulation in IgE-activated rat basophilic leukemia cell line (RBL-2H3) cells. We investigated the regulatory effects of tricin and ETZL on degranulation, production of cytokines and lipid mediators, and signaling proteins involved in the IgE-bound high-affinity IgE receptor activation, mitogen-activated protein kinase, arachidonic acid and Syk. The production of β–hexosaminidase, tumor necrosis factor-α, interleukin-4, leukotrienes (LT) B4, LTC4 and prostaglandin E2 in IgE-stimulated RBL-2H3 cells were significantly inhibited by exposure to tricin or ETZL. Moreover, tricin and ETZL inhibit the phosphorylation of cytosolic phospholipase A2, 5-lipoxygenase and cyclooxygenase-2. Furthermore, the phosphorylation of Akt, ERK, p38, JNK, protein kinase Cδ and phospholipase Cγ1 were effectively suppressed by both samples. Exposure to tricin or ETZL also significantly decreases the phosphorylation of Lyn and Syk, but has minimal effect on Fyn. Taken together, our data indicate that tricin and ETZL are potential anti-allergic materials that could be applied for the prevention of allergy-related diseases.Tricin, a flavone present in rice bran, is confirmed as the major efficacious compound present in the enzyme-treated  Zizania latifolia (Gramineae) is the only member of the wild rice genus Zizania native to Asia, including China, Korea and Japan. Z. latifolia Turcz differs botanically from the other wild rice species, including Z. aquatica, Z. palustris and Z. texana. The Z. latifolia grains impart numerous pharmacological effects, including suppression of hyperlipidemia [Z. latifolia exhibits a variety of inhibitory activities, such as H2O2-induced apoptosis in Neuro2A cells [ipidemia , reductiipidemia , decreasipidemia , improveipidemia  and antiipidemia . Recentl2A cells , angiote2A cells  and ultr2A cells ,7. The m2A cells .Z. latifolia [2 (PGE2) production in colon cells and murine plasma [Z. latifolia. Treatment with an enzyme mixture significantly increased the tricin content in a hydrolyzed ethanol extract [In our previous study, we identified five derivatives of tricin   in the matifolia . The trie plasma . Tricin 4 and LTC4 attract neutrophils and stimulate the late-phase allergic reaction [2) response  activate B cells to produce IgE, thereby activating mast cells and inducing allergic inflammation [Mast cells play a major role in IgE-mediated allergic responses (hypersensitivity) by producing inflammatory mediators such as histamine, leukotrienes, prostaglandins and cytokines upon binding of an antigen to the IgE-bound high-affinity IgE receptor (FcεRI) . Since hreaction . Cytokinammation .2), 5-lipoxygenase (5-LO) and COX-2, and secretions of leukotrienes and prostaglandins [In addition, FcεRI stimulation results in the immediate activation of two Src family kinases, Lyn and Fyn, and a tyrosine kinase spleen tyrosine kinase (Syk), to initiate mast cell degranulation, with subsequent activation of the phospholipase Cγ (PLCγ) and protein kinase B (Akt) signaling pathways ,13. The glandins ,12. Phosglandins . TherefoAntihistamines and mast cell stabilizers are widely used as anti-allergy agents for ameliorating allergic inflammatory symptoms, despite some side-effects such as drowsiness. Recently, efficacious edible plant materials devoid of any side effects are gaining attention as safe anti-allergic agents. RBL-2H3 cells express FcεRI on the cell membrane, and the IgE-bound FcεRI can be activated by antigens; thus, these cells are widely used for in vitro investigations on the mechanism of mast cell activation ,17.Z. latifolia extract (ETZL) remain unclear. Therefore, to understand the mechanism by which tricin or ETZL alleviates the allergic response in IgE-mediated allergic reactions in RBL-2H3 cells, this study investigated the regulatory effects on signaling cascades, such as FcεRI, arachidonic acid and MAPKs.Mechanisms associated with the anti-allergic action of tricin isolated from enzyme-treated Z. latifolia exerts excellent anti-degranulation activity in RBL-2H3 cells, and contains tricin and its derivatives as the major components for anti-allergic activity. The identified derivatives include tricin-7-O-β-d-glucopyranose, tricin-4’-O-(threo-β-guaiacylglyceryl) ether 7-O-β-d-glucopyranose, tricin-4’-O-(erythro-β-guaiacylglyceryl) ether 7-O-β-d-glucopyranose, tricin-4’-O-(threo-β-guaiacylglyceryl) ether 7’’-O-β-d-glucopyranose and tricin-4’-O-(erythro-β-guaiacylglyceryl) ether 7’’-O-β-d-glucopyranose [Z. latifolia (data not published). Additionally, an enzymatic treatment applied to augment the amount of tricin increased the content by 1.27-fold in ETZL as compared to non-enzyme treated ethanol extract (NEZL), giving 25.0 mg/100 g dried Z. latifolia with extraction yield of 17.45%, estimating 0.14% tricin in ETZL [Z. latifolia (ETZL). As reported in our previous studies, the methanol extract from aerial pyranose ,18. The  in ETZL . Our prep > 0.05). Effects on the IgE-mediated degranulation were then evaluated by determining the activity of β-hexosaminidase, a biomarker of histamine release, in IgE-activated RBL-2H3 cells. As expected, NEZL exhibit lower inhibitory activity against the release of β-hexosaminidase (50% inhibitory concentration (IC50), 106.79 μg/mL), as compared to ETZL  (p < 0.001)  as antigen. The effects of NEZL, ETZL and tricin were determined on the cell viability by the MTT assay. As shown in < 0.001) B. In add84 ng/mL B. These Actinidia arguta extract has been recognized as a functional ingredient in facilitating the prevention of atopy, food allergy and asthma [Monitoring of mast cell degranulation using the β-hexosaminidase assay in RBL-2H3 cells is widely used to determine the effectiveness on anti-allergic responses, and is a convenient in vitro cell model for studying new molecules that inhibit mast cell activation or degranulation ,20. Histd asthma ,24,25.p < 0.001), respectively. The TNF-α production was inhibited after exposure to tricin  and ETZL  in dose-dependent manners (p < 0.001) was higher than tricin (p < 0.01) at 100 µg/mL, indicating that apart from tricin, ETZL may have other bioactive compounds imparting the anti-allergic inflammation, including derivatives of tricin and other trace amounts, as shown in our previous reports [Following the immediate histamine release subsequent to antigen-mediated FcεRI aggregation, degranulation of mast cells results in the secretion of inflammatory mediators, including TNF-α, IL-4, leukotrienes and prostaglandins, which play key roles during the late phase of inflammatory reactions ,17. Inhi manners A. Simila manners B. Simila2 cell differentiation, which subsequently causes anaphylaxis [The histamine released from mast cells increases the expression level of FcεRI on mast cells, thereby stimulating the cells for further antigen-induced activation, resulting in the production of inflammatory cytokines and lipid mediators. Recent studies have determined that apart from early-phase allergic responses, mast cells also contribute to the late-phase reactions by modulation of cytokines and mediators . TNF-α, phylaxis . Severalphylaxis , quercetphylaxis , luteoliphylaxis , chalconphylaxis  and curcphylaxis , inhibit4, LTC4 and PGE2, which initiate chronic inflammation in allergy-related diseases [4, LTC4 and PGE2. Preincubation with varying concentrations of tricin or ETZL prior to antigen stimulation markedly suppresses the production of LTB4 , LTC4  and PGE2  (p < 0.001)  A–C. Thes2, 5-LO and COX-2, in IgE-activated RBL-2H3 cells. Both samples inhibit the phosphorylation of cPLA2, 5-LO and COX-2, as seen in We further investigated the effects of tricin and ETZL on the activation of enzymes involved in the synthesis of eicosanoids, including cPLA2 generates arachidonic acid (a potent metabolic precursor) from phospholipid; subsequently, the arachidonic acid metabolizing enzymes (COX-2 and 5-LO) produce PGE2 and LTs, respectively, from arachidonic acid. Numerous studies have reported that various compounds  are natural inhibitors of arachidonic acid metabolite formation and the metabolite-mediated allergic inflammation [Oryza sativa L. is also reported as an anti-inflammatory molecule for COX-2-mediated PGE2 production in LPS-stimulated human peripheral blood mononuclear cells [MinApc mice (in vivo) [Phosphorylation of cPLAammation . Tricin ar cells  as well in vivo) . Our dat2 to synthesize arachidonic acid metabolites by 5-LO or COX-2, inducing the secretions of LTB4, LTC4 or PGE2. Akt is a downstream effector of Syk, and the phosphorylation of Akt is involved in mast cell degranulation [4, LTC4 and PGE2). Since activation of MAPKs plays major roles in the formation of inflammatory cytokines, we next examined the effects of tricin and ETZL on the Akt and MAPK signaling pathways in IgE-stimulated RBL-2H3 cells . The phonulation . Protein2+ mobilization, which is a crucial step for mast cell degranulation [Upon binding of an antigen to the IgE-bound FcεRI, the signaling pathways involving PI3K/PLCγ/protein kinase C (PKC) and MAPKs are activated. The phosphorylation of PLCγ1 and PKCδ stimulates the secretion of histamine along with β-hexosaminidase and inflammatory cytokines, resulting in allergic reactions ,31. The nulation ,33. ThusTo investigate the regulatory effects of tricin and ETZL on the early stage of FcεRI signaling pathway, we next examined the phosphorylation levels of Fyn, Lyn and Syk in IgE-activated RBL-2H3 cells. The phosphorylation levels of Lyn and Syk were up-regulated by IgE-stimulation , but Fyn levels were minimally altered C,D. TricPlatycodon grandiflorum and Loranthus parasiticus extract have been reported to inhibit allergic reactions by suppressing the phosphorylation of Akt, ERK, p38 and JNK, as well as inhibiting Syk and Lyn in antigen-stimulated RBL-2H3 cells, suggesting possible therapeutic applications in allergy related diseases [Among the numerous natural resources known to inhibit Syk and MAPK activation in mast cells, saponins obtained from roots of diseases ,34.4, LTC4 and PGE2 by affecting the enzyme activities of 5-LO and COX-2 as well as ERK and cPLA. Furthermore, our data demonstrates that phosphorylation of Lyn and Syk proteins involved in the early stage of FcεRI signaling pathway are effectively suppressed by tricin. In the current study, the numerous reactions that inhibit allergic responses after exposure to tricin were investigated at various phosphorylation steps in the signaling pathways triggered by antigen bound IgE-FcεRI on mast cells. Further studies are required to determine the in vivo efficacy of tricin containing ETZL, using allergy animal models for asthma, allergic rhinitis and atopic dermatitis. Overall, our data confirms that tricin and ETZL possess potential anti-allergic activity, and tricin can be used as a phytochemical to inhibit the mast cell-derived mediator release, in the development of functional products related to allergic disorders.In conclusion, the present study demonstrates that ETZL exerts anti-allergic effects in IgE-stimulated mast cells, and tricin is responsible for its preventive action against the allergic reactions. The suppressive mechanism of tricin involved in the signaling cascade necessary for IgE-mediated allergic reactions, were investigated via the FcεRI signaling pathway related to mast cell degranulation and secretions of cytokines and chemo-attractants. Tricin inhibits the release of β-hexosaminidase, production of proinflammatory cytokines TNF-α and IL-4 through the inhibition of PLCγ/PKCδ and MAPKs pathways, and also the synthesis of arachidonate metabolites LTBN-acetyl-β-d-glucosaminide (p-NAG) were purchased from Sigma Aldrich Co. . Antibodies against phospho-protein kinase B , phospho-cPLA2 (#2831), phospho-extracellular signal-regulated kinase 1/2 , phospho-c-Jun N terminal kinase 1/2 , phospho-Src family protein kinase , phospho-p38 (#9211), phospho-PKCδ (#2055), phospho-PLCγ1 (#2821), phospho-spleen tyrosine kinase , horseradish peroxidase-linked anti-rabbit secondary antibody (#7074) and anti-mouse secondary antibody (#7076) were purchased from Cell Signaling Technology . Specific antibodies against phospho-Fyn (orb128087) were obtained from Biorbyt Ltd. . A specific antibody against 5-LO (#10007820) and EIA kits for LTC4, LTB4 and PGE2 were obtained from Cayman Chemical Co. . ELISA kits for IL-4 and TNF-α were purchased from R&D systems, Inc. . Minimum Eagle’s medium (MEM) and fetal bovine serum (FBS) were from the American Type Culture Collection . All other chemicals and reagents used were of analytical grade.3-2-,5-diphenyltetrazolium bromide (MTT), dinitrophenyl-human serum albumin (DNP-HSA), anti-DNP-IgE and 4-nitrophenyl Z. latifolia were purchased from the Pureunsan Agricultural Association Corporation  and ETZL was provided by the BTC Corporation . Briefly, the dried leaves of Z. latifolia were incubated with mixed hydrolysis enzymes  at 35 °C for 16 h in H2O, followed by heating to inactivate the enzymes. The extracted solution was filtered and acquired. After extraction of the residue with 70% ethanol  at 80 °C for 6 h, the filtered and extracted solution was mixed with the first enzyme extract and the total enzyme extract was concentrated and dried to produce ETZL. Tricin was isolated from ETZL as previously described [The aerial parts of escribed . The isov/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, at 37 °C in a humidified atmosphere of 5% CO2. All experiments contain a control group as the vehicle control group, comprising 0.1% DMSO.RBL-2H3 cells, a mast cell line originating from rat basophilic leukemia, were cultured in MEM medium supplemented with 10% (5 cells/well) in MEM medium supplemented with 10% FBS, and cultured overnight at 37 °C. The cells were washed with 1 × PBS, and subsequently incubated with anti-DNP-IgE (0.05 μg/mL) for 24 h. The IgE-sensitized cells were incubated with varying concentrations of tricin or ETZL  for 1 h. DNP-HSA (0.1 μg/mL) was then added to the wells, and the mixture was incubated for a further 4 h. To measure cell viability, the absorbance was measured at 550 nm using a well-plate multi-reader.Briefly, the RBL-2H3 cells were seeded in a 24-well plate (1 × 105 cells/well), overnight at 37 °C. Cultured cells were washed with 1 × PBS, and subsequently incubated with anti-DNP-IgE for 24 h. IgE-sensitized cells were exposed to varying concentrations of tricin or ETZL  for 1 h, spiked with DNP-HSA and then incubated for a further 4 h. To measure the amount of β-hexosaminidase activity released from the cells, the culture medium was transferred and centrifuged  at 4 °C. The supernatant (25 μL) was mixed with 50 μL p-NAG (10 mM) in 0.1 M sodium citrate buffer (pH 4.5) in a 96-well plate, and then incubated for 1 h at 37 °C. The reaction was terminated by adding sodium carbonate buffer . The β-hexosaminidase activity was determined by measuring the difference in absorbance at 405 nm.The RBL-2H3 cells were incubated in a 24-well plate (1 × 10g for 10 min) at 4 °C, and the samples were stored at −80 °C until use. The concentrations of IL-4 and TNF-α were determined using ELISA kits , according to the manufacturer’s instructions. To determine the levels of LTB4, LTC4 and PGE2 all culture media were centrifuged  at 4 °C, and the supernatant was stored at −80 °C until use. The concentrations of LTB4, LTC4 and PGE2 were determined using EIA kits , according to the manufacturer’s instructions.To measure the cytokine levels in the culture medium, all culture media were centrifuged  containing protease inhibitor  and phosphatase inhibitor , with subsequent incubation on ice for 30 min. Protein concentrations in cell lysates were determined using a BCA protein assay . Proteins (50 μg) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes , blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and then incubated with primary antibodies for 16 h at 4 °C. After three washes in Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with horseradish peroxidase-linked secondary antibodies  for 1 h. Proteins were detected by enhanced chemiluminescence, and visualized using image software . RBL-2H3 mast cells were lysed in lysis buffer (Pro-Prept test, and a significant difference is considered at p < 0.05, p < 0.01 and p < 0.001.Data are presented as means ± SD values of three experiments. Differences among groups were examined using Student‘s


Post-translational modification of proteins, such as tyrosine phosphorylation, plays a major role in driving the oncogenic activity of oncogenes. WAVE3 (WASF3), an adaptor and actin cytoskeleton remodeling protein, contributes to cell migration, cancer cell invasion, and metastasis. WAVE3 plays a vital role in the progression and metastasis of triple negative breast cancer (TNBC), in part through the regulation of cancer stem cells (CSCs). Several studies have shown that WAVE3 tyrosine phosphorylation is required for its oncogenic activity. Moreover, our recent study showed that the proline rich domain (PRD) of WAVE3 is required for maintenance of the CSC niche in breast cancer by regulating the nuclear translocation of the CSC-specific nuclear transcription factor YB1. Here, we show that the PRD domain of WAVE3 and its phosphorylation are essential for driving the oncogenic activity of WAVE3. We show that phosphorylation of WAVE3 PRD is essential for migration and invasion of breast cancer cells in vitro, as well as tumor growth and metastasis in vivo. Mechanistically, we show that phosphorylation of the WAVE3 PRD is essential for interaction between WAVE3 and YB1. Loss of PRD phosphorylation inhibits such interaction and the YB1-mediated activation of expression of CSC markers, as well as the WAVE3 mediated activation of EMT. Together, our study identifies a novel role of WAVE3 and its PRD domain in the regulation of the invasion metastasis cascade in BC that is independent of the known function of WAVE3 as an actin cytoskeleton remodeling protein through the WAVE regulatory complex (WRC). Metastasis also accounts for more than 90% of cancer-related deaths2. WAVE3 (WASF3) is a member of the Wiskott-Aldrich Syndrome Protein (WASP) family that was originally identified as regulator of the actin cytoskeleton remodeling4. Several other studies have also implicated WAVE3 in several aspects of cell migration, cancer cell invasion and tumor metastasis  an N-terminal basic region (BR), (ii) a central proline-rich domain (PRD) which is known to play an important role for the enhancement of Cdc42-WASP-induced nucleation of actin polymerization, and (iii) a C-terminal Verprolin homology, Cofilin homology and Acidic (VCA) domain, that binds to the Arp2/3 complex and initiates the growth of actin filaments required for cell motility and migration8. Like WAVE1 (WASF1) and WAVE2 (WASF2), the function of WAVE3 in actin polymerization and actin cytoskeleton remodeling is regulated through its inclusion in the WAVE regulatory complex (WRC)9. WRC consists of five protein subunits: PIR121, Nap125, HSPC300, and Abi1, where WAVE3 is kept in an inactive state through the formation of a complex with the other four protein subunits of the WRC10. Subsequent recruitment of Rho GTPase Rac1 to the WRC releases WAVE3 from this complex11, which becomes free to bind to the actin nucleator Arp2/3 complex and activate actin polymerization13. Recent studies from our group have however identified a function for WAVE3 that may be independent of its traditional relationship with the WRC, Arp2/3 complex and actin polymerization15.Cancer metastasis is a complex process that allows tumor cells to escape the primary site and colonize distant organs via the blood circulation or the lymphatic system17. Phosphorylation of WAVE3 at four tyrosine residues  is mediated by the non-receptor tyrosine kinase c-Abl, downstream of PI3K16. A more recent study has shown that the oncogenic activity of WAVE3 in BC is significantly enhanced as a result of tyrosine phosphorylation15. Two of the four tyrosine residues of WAVE3, Y248 and Y337, reside within the PRD domain. The PRD domain of WAVE3 was also shown to be necessary for the translocation from the cytoplasm to the nucleus of the transcription factor YB1, and the subsequent activation of cancer stem cells (CSC)-specific transcription factors, such as Nanog and Oct4 and Sox14. This novel function of WAVE3, that is mediated by the PRD domain, seems to be independent of the WRC and actin polymerization20. In this study, we show that phosphorylation of WAVE3 PRD domain is required for cell migration, invasion, as well as the maintenance of the cancer stem cell phenotype in TNBC. We also show that loss of phosphorylation of WAVE3 at the PRD domain suppresses the epithelial-to-mesenchymal (EMT) programs, as well as significantly inhibits expression of CSC-specific markers through the dysregulation of the WAVE3-YB1 interaction.Post-translational modifications (PTMs) of proteins plays a major role in driving the oncogenic activity in several cancer types including BC. Several studies have shown that PTM of WAVE3 through its tyrosine phosphorylation was critical for that WAVE3-mediated regulation of tumor progression and metastasis of TNBCOur data demonstrates that the oncogenic activity of WAVE3 can be driven by phosphorylation of its PRD domain, and targeting this PTM may serve as a potential therapeutic option for treating patients with TNBCs, thus preventing cancer progression and metastasis.All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee and conducted in accordance with the guidelines and regulations set and approved by the MetroHealth Medical Center, Case Western Reserve University, and NIH. For this study, we used six- to eight-weeks-old female NSG .15. For each BC cell line, we used two different and verified WAVE3-specific sgRNAs and a scrambled sgRNA i.e., nonsilencing sgRNA15. We used western blot to verify the loss of WAVE3 expression. For stimulation of cells with PDGF, cells were serum starved in serum-free DMEM without antibiotics overnight. Next day, the cells were stimulated with 100 ng/ml PDGF (Millipore) for 10 min. The PI3K inhibitor LY294002 was obtained from SelleckChem and used at a concentration of 10 µM for 4 h.MDA-MB-231 and 4T1 TNBC cell lines were obtained from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. We used STR DNA fingerprinting analysis for regular authentication of cells that we used for this study. WAVE3-deficient (WAVE3-KO) cells were produced using lentiviral transduction as describedRabbit anti-WAVE3, rabbit anti-YB1, mouse anti-phosphotyrosine (PY20) primary antibodies were obtained from Cell Signaling Technology (CST) and used at a dilution of 1:1,000. Mouse anti-GFP was obtained from Clontech Laboratories, Inc. and used at a dilution of 1:5000. Goat horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Calbiochem, Sigma-Aldrich and used at a dilution of 1:2000. Mouse monoclonal anti-β-Actin was obtained from Sigma-Aldrich and used at a dilution of 1:5000. Mouse anti-E-cadherin antibody (1:200) was from BD Transduction Laboratories and mouse anti-Vimentin antibody (1:200) was from Santa Cruz Biotechnology, Inc. Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:500) and Hoechst 33,342 (1:1000) were from Invitrogen. Gel electrophoresis reagents were obtained from Bio-Rad. All the qRT-PCR primers were obtained from Integrated DNA Technologies (IDT) and are listed in Table 22. Cells were grown in 6 well plates and washed twice with ice-cold PBS. Immediately, ice-cold RIPA lysis buffer in the presence of a mixture of proteases and phosphatase inhibitors. Cells were rapidly scraped off the plates, and the crude lysates were transferred to prechilled tubes and centrifuged at 15,000 RPM for 20 min at 4 °C. For immunoblotting, cell lysates containing equivalent amounts of total protein (25 μg) were supplemented with SDS sample buffer and resolved on a 10% SDS–polyacrylamide gel, followed by transfer to PVDF membranes using Bio-Rad gel and transfer apparatus. Membranes were incubated in 5% bovine serum albumin (BSA) for 1 h at room temperature, washed with PBS, followed by incubation with the primary antibody overnight at 4 °C. Membranes were then washed and incubated in the appropriate secondary antibody at room temperature for 1 h, and the signals were developed using a Pierce ECL Western blot chemiluminescence detection kit and visualized with ChemiDoc MP Imaging System. For signal quantification, ImageJ software was used according to the parameters described in ImageJ user guide (http://rsbweb.nih.gov/ij/docs/guige/146.html). Average values from 3 different blots are presented.Immunoblotting analyses were performed as previously described4) were seeded into six well-plates in complete growth medium, then harvested at one day intervals over 5 days, and counted using a hemocytometer as previously described25. Cell viability was assessed using trypan blue staining. For wound-healing assay, cells were grown to confluency and a scratch was made by dragging a thin pipet tip across the cell surface. Cells were washed with serum-free medium to remove detached cells and then incubated in IncuCyte Live-Cell Analysis System. Images were taken every 24 h using the same system. Wound closure was quantified by measuring the width of the remaining wound using ImageJ 1.8.0 (NIH). Assays were performed in triplicates, and the values plotted were the average of three independent experiments.Cell proliferation was also assessed by counting the number of viable cells. Cells  on top. The plate was then placed inside the IncuCyte Live-Cell Analysis System and scanning was scheduled every 6 h continuously for 10 days.MDA-MB-231, 4T1 or their derivatives  were seeded in 96-well ultra-low attachment (ULA) plate and centrifuged at 125×23.For multi-tumorsphere formation, a 96-well ULA plate was coated with 40 μl Matrigel (in complete culture media) and placed in the incubator for 30 min to allow polymerization. Next, cells  were plated on top of the layer of Matrigel and placed inside the IncuCyte Live-Cell Analysis System and scanned every 6 h continuously for 10 days to monitor multi-spheroid formationTumorspheres, grown as described above, were transferred to a 12-well plate containing 400 µL PBS and the tumorspheres were allowed to settle in bottom of the plate for 3 min before the plate was tilted slightly and PBS removed by gentile aspiration making sure the tumorspheres are not disturbed. Tumorspheres were then fixed for 20 min at room temperature in 3.8% formalin in PBS, and permeabilized in 0.025% w/v Triton X-100 for 5 min at room temperature, followed by three washes in PBS for 3 min each. Next, tumorspheres were blocked in 5% donkey serum in PBS for 30 min and washed 3 times in PBS, then incubated overnight with the primary antibody (1:200 dilution) in 5% DS at 4 °C. Tumorspheres were carefully washed 3 times and next incubated with Alexa Fluor 594-conjugated donkey anti-mouse IgG secondary antibody (1:500 dilution) for 1 h at room temperature, followed by three washes in PBS. Nuclei were stained with Hoechst 33,342 (1:1000 dilution in PBS) for 10 min and washed 3 times. Finally, tumorspheres were observed and documented under the Leica DMi8 microscope.n = 5) were implanted into the mammary fat pads of six- to eight-weeks-old female NSG mice. Each mouse was implanted on both the left and right abdominal mammary fat pad. Mice were anaesthetized using isofluorane prior to injecting tumor cells. Tumor growth was monitored twice every week and the tumor volume was measured using Instant Readout Digital Caliper . At the endpoint, tumors were excised, weighed and fixed with 4% paraformaldehyde. For the experimental metastasis assay, cells (2 × 106) suspended in 0.15 ml of sterile PBS were injected using a 28-gauge needle into a tail vein of six- to eight-weeks-old female NSG mice. Mice were sacrificed 5 weeks later, and the recovered lungs were fixed with 4% paraformaldehyde. Lung metastasis nodules were counted, and the results were plotted as average number of foci per lobe. Metastasis was also quantified by qt-PCR of human genomic DNA from total DNA of mouse lungs using human DNA specific primers as described25. PCR primers and their sequences are listed in Table In the spontaneous metastasis assay, parental (GFP), WAVE3-deficient (W3-KO), WAVE3-deficient overexpressing wild-type WAVE3-PRD-GFP fusion (PRD-WT) or phospho-mutant WAVE3-PRD-GFP fusion (PRD-DM) MDA-MB-231 cells  in- frame with GFP in the pEGFP-C2 expression vector (Clontech). The resulting plasmid construct was used as a template for site-directed mutagenesis to introduce the Y to F mutations using the primers listed in Table 16. The GFP-recombinant vector or the empty GFP expression control vector was used for stable transfections using standard protocols. The correct size of the fusion proteins was verified by Western blot analysis. Oligonucleotide primers used for site-directed mutagenesis and sequencing were from Qiagen and are described in Table 16.GFP-tagged WAVE3 PRD constructs were generated as previously described26. Briefly, cells were lysed in TRIzol reagent (Invitrogen), and total RNA was extracted according to the manufacturer’s instructions. Total RNA, resuspended in RNase-free water, was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific); and 1 μg of RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis System RT-PCR kit (Invitrogen). The resulting cDNA was used a template for qtRT-PCR as described previously13, using the C1000 Touch Thermal Cycler CFX96 Real-Time System (Bio-Rad). Oligonucleotide primers used for qtRT-PCR were from Qiagen and are listed in Table RNA extraction was performed as previously describedp-value < 0.05 was considered statistically significant. For statistical analysis of qRT-PCR data, logarithmic values were converted to ΔΔCt values.All the experiments were performed in triplicates. Data are presented as mean and S.D., and a two-tailed Student’s t-test was used to compare the samples assuming equal variances for the two populations. A 32. Our published studies have also shown that phosphorylation of WAVE3 at four tyrosine residues  are located within the PRD domain of WAVE3  WAVE3 can be detected at basal levels in MDA-MB-231 . Our recently published study has shown that WAVE3 phosphorylation is required for the activation of BC cell migration and tumorsphere growth and invasion15. Whether the PRD domain of WAVE3 and its phosphorylation are also important in such oncogenic activities have not been reported. Parental MDA-MB-231 or 4T1 (GFP), their W3-KO derivatives expressing either GFP, wild-type PRD or phospho-mutant PRD were subjected to 2-dimensional (2D) wound-healing assay and the extent of the wound that remained open at the end of the experiment was compared between the experimental groups. In the parental (GFP) MDA-MB-231 cells less than 5% of the wound remained open, while in the W3-KO cells more than 70% of wound remained open  mice were inoculated with parental MDA-MB-231 cells (GFP), W3-KO, or W3-KO re-expressing either wild-type PRD domain (W3-WT-PRD) or phospho-mutant PRD domain (W3-PRD-DM), and tumor growth was assessed over 8 weeks. Loss of WAVE3 inhibited the growth of primary tumors Fig. A,B. Re-eNext, we sought to investigate how the loss of phosphorylation of the WAVE3-PRD domain influences BC metastasis. In the spontaneous metastasis assay, after the primary tumors were excised from the mammary gland sites, the mice were sacrificed, lungs removed, and lung metastasis nodules counted. We found the number of lung nodules (metastasis foci), was reduced by more than tenfold (p < 0.01) in mice implanted with the WAVE3-deficient (W3-KO) cells compared to their parental (GFP) counterparts Fig. D–G. In tWe also used the lung colonization  assay, in which cancer cells were injected directly in the blood via the lateral tail vein to determine the effect of loss of phosphorylation of the WAVE3-PRD domain on metastasis. Here again Fig. H–K we fo37. Our published studies showed that the interaction between WAVE3 and YB1 is required for the translocation of YB1 to the nucleus to allow YB1 to exert its transcription factor function14. We have also shown that interaction between YB1 and the PRD of WAVE3 is essential for the nuclear translocation of YB1 and for the regulation of transcription of the CSCs-specific genes14. Here, we sought to investigate whether phosphorylation of WAVE3 and its PRD domain are required for the interaction with YB1. First, we used co-immunoprecipitation of cell lysates from WAVE3-deficient MDA-MB-231 cells that stably express either wild-type WAVE3 (W3-WT) or phospho-mutant WAVE3 (W3-Y4F) to show that when all four tyrosine residues are mutated to unphosphorylated phenylalanine (W3-Y4F)16, the interaction between WAVE3 and YB1 is completely lost  results in a significant (p < 0.05) decrease in expression levels of EMT markers Vimentin  in MDA-MB-231 cells resulted in a significant (p < 0.05) inhibition of Nanog  is required for the phosphorylation of WAVE3 PRD domain. We presented evidence that phosphorylation of WAVE3 at both tyrosine residues Y248 and Y337 can be attained by PDGF downstream of PI3K. In fact, the activation of the PI3K by PDGF is not direct, but instead is mediated downstream of PDGFR42. Inhibition of WAVE3 phosphorylation by LY294002 treatment is in fact mediated through the inhibition of PDGFR, which in turn results in inhibition PI3K and its downstream effectors, such as the c-Abl non-receptor tyrosine kinase, which we showed is directly involved in WAVE3 phosphorylation16. Several studies have shown that, in addition to PI3K/c-Abl, WAVE3 phosphorylation can also be achieved downstream of TGF-β and EGF15 as well as JAK1/217. WASP, a WAVE3-related protein was also shown to be phosphorylated by members of the Src family kinases43, while N-WASP can be activated downstream of tyrosine kinases in a PIP2-depedent manner44. On the other hand, WAVE1 (WASF1) can be activated through its phosphorylation by MAP kinases45. Whether WAVE3 can be phosphorylated by this family of kinases is however not known. We are, however, very confident that the signaling axis of PI3K is a major activator of WAVE3 phosphorylation, since treatment of PDGF-stimulated MDA-MB-231 cells with the PI3Kα-specific inhibitor alpelisib46, almost completely abrogated WAVE3 tyrosine phosphorylation15.In this study, we also demonstrated that phosphorylation of WAVE3 PRD domain is required for the activation of the CSC phenotype and the EMT programs. Our previous studies have reported that WAVE3 phosphorylation is required for the activation of cell migration, tumorsphere growth and invasion in TNBC47 and  complex to activate elongation of actin filaments47. The BR, which is ~ 200 amino acid long is loosely conserved between the three members of the WAVE family, and its specific function is still largely unknown, expect for its speculated auto-inhibitory effect on the WAVE regulatory complex (WRC), where it was postulated that folding of this BR domain masks the binding of RAC-GTP to WAVE3 that is required for the activation of the WRC48. The PRD domain is also not very well conserved among the WAVE family49. More importantly, the two tyrosine residues Y248 and Y337 are unique to the PRD domain of WAVE3 and are conserved in both the human and the mouse WAVE3 proteins, but not found in the other WASP/WASF family members47, which makes the function of this domain specific to WAVE3. The Y-box-binding protein-1 (YB1) has a dual function. In the cytoplasm, YB1 acts as a regulator mRNA translation and stability, while in the nucleus YB1 functions as a transcription factor that regulates expression of several CSC-specific genes52. Our previous studies showed that interaction between YB1 and WAVE3 is required for the translocation of YB1 from cytoplasm to the nucleus, since YB1 does not have a nuclear translocation signal. In this instance WAVE3 plays the role of a shuttle protein that transports YB1 inside the nucleus to allow it to perform its nuclear transcription function and activation of expression of the CSC-specific transcription factors, Oct4 and Nanog and Sox14. Accordingly, we found WAVE3 to play a critical role in the maintenance of CSC niche through its interaction with YB114. Here, we showed that expression of the CSC-specific transcription factors, Oct4 and Nanog, is significantly diminished as a result of loss of expression of WAVE3 or phosphorylation of its PRD domain, therefore, demonstrating the requirement of PRD and its phosphorylation in the regulation of CSC maintenance. Finally, it has now been widely demonstrated that the oncogenic activity of WAVE3 is closely associated with numerous hallmarks of cancer, including EMT (Reviewed in5). Our results found that loss of WAVE3 expression or loss of phosphorylation of its PRD domain inhibit the EMT programs through downregulation of vimentin, N-cadherin, ZEB1 and ZEB2, and upregulation of E-cadherin. In addition, the miR200 family are known master regulators of EMT57. Our previous studies have shown that the WAVE3-mediated modulation of the EMT programs is regulated downstream of the miR200 family28. While the studies by Burk et al.53 and Korpal et al.57 have identified a negative feed-back loop between ZEB1/ZEB2 and members of the miR200 family that regulates EMT and cancer cell invasion, while the report by Teng and colleagues have shown that WASF3 (WAVE3) regulates the ZEB1-mediated inhibition of miR-200, and the subsequent activation of BC invasion cells29. The involvement of the phosphorylated WAVE3 and its PRD domain in the regulation of ZEB1 and ZEB2 further cements the major role that WAVE3 plays in the very complex biological process such as EMT. Together, our findings show that phosphorylation of WAVE3 PRD domain is important for invasion and metastasis of TNBC tumors, and is required maintenance of CSC population. Therefore, targeted inhibition of phosphorylation of the PRD domain of WAVE3 may represent a novel therapeutic option for the inhibition of TNBC progression and metastasis.Using 2D wound-healing assay and 3D tumorsphere growth and invasion assays, we also showed that phosphorylation of the WAVE3 PRD domain enhances tumor growth and metastasis, while loss of phosphorylation in the phospho-mutant WAVE3 PRD domain inhibited these oncogenic properties. WAVE3 consists mainly of three distinct subdomains: the N-terminus Basic Region (BR), a central proline-rich domain (PRD) and the distal Verprolin Cofilin Acidic (VCA) domainand Fig. A. The VCSupplementary Information 1.Supplementary Information 2.


The radiative effect on microphysics (REM) plays an important role in the dew/frost formation near the surface. How REM impacts cirrus clouds is investigated in this study, using bin microphysical model simulations and coincident data of the CloudSat and Global Precipitation Measurement (GPM) satellites. REM affects ice crystal spectrum with two types: radiative cooling and warming. Radiative cooling, as predicted by the bin‐model simulations, favors the formation of horizontally oriented ice crystals (HOICs), but radiative warming does not. Hence, a test of REM can be transformed to a test of HOICs, because HOICs can be measured by the microwave polarization observations of the GPM Microwave Imager (GMI) at 166 GHz. To analyze the GMI data for their HOIC distribution, clouds are sorted into four groups with different optical depth and altitude, based on the radiative cooling/warming ratio (or eta) computed with satellite‐retrieved ice water content. Their HOIC distributions  agree well with those predicted by the bin‐model simulations. The general agreement between the GMI observations and bin‐model simulations suggests that REM is common in cirrus clouds and impacts cirrus clouds significantly. A microphysical bin model is used to simulate the radiative effect on microphysics (REM) at different altitudesThe radiative factor of REM is calculated using the satellite‐retrieved ice water content and other variablesModel results show that REM is stronger in the midlevel thick clouds than in the high‐level ones Instead, it is the effect of radiation on cloud particle spectrum via a radiation‐induced difference in temperature between air and cloud particles see Appendix ppendix AREM in clouds is usually noticed by observing its consequence on cloud particles. Stephens , for exaSince REM varies greatly in space and time, REM needs to be classified first for a comparison to observations Zeng, . REM is σ is the Stefan–Boltzmann constant, T is air temperature, and F+ and F− are the upward and downward fluxes of infrared radiation (IR) in the atmosphere, respectively. Roughly speaking, ηz is a ratio of total IR warming (numerator) to IR cooling (denominator) of a horizontally oriented plate‐like body , ηz and thus REM vary from cloud to cloud.where body see ppendix Aηz < 1, ice crystals undergo radiative cooling and thus their spectrum is broadened; otherwise, ice crystals undergo radiative warming and their spectrum is narrowed  satellite measures convection and thick clouds via the GPM Microwave Imager (GMI), providing information on the horizontal structure of thick clouds  nadir‐looking radar to measure clouds, ranging from optically thin clouds to deep convection . Thus, an interesting question is why the midlevel clouds correspond to high 166PD, whereas the high‐level clouds correspond to low 166PD, which is discussed next with REM.2.3ηz < 1) that in turn brings about HOICs there via REM , its high value usually corresponds to an abundance of HOICs near cloud top , liquid water content (LWC), and other ancillary atmospheric variables.In situ The retrieved IWC comes from the 2C‐ICE (CloudSat and CALIPSO Ice Cloud Property) product that combines the data of CloudSat radar and CALISPO lidar  that is based on the CloudSat radar observations  besides ηz bring about the difference in 166PD between Clouds A and C, which are addressed next using REM modeling.However, thick cloud C as well as its neighboring clouds between 220 and 350 km brings about a relatively low 166PD, although 3ηz and cloud height. In this section, the microphysical bin model of Zeng , replicating the interaction between ηz and other processes (including clouds).In the bin model, ηz represents the inward radiative flux through parcel boundary normalized by a scale of energy flux σT4 (or the blackbody irradiance at temperature T), its magnitude determines the radiative energy budget of each ice crystal in the parcel, although the energy budget of each ice crystal depends on other factors such as ice crystal shape, size, and orientation , its growth rate is larger than that without radiation; conversely, when ηz > 1 (radiative warming), its growth rate is lower than that without radiation.For an ice crystal in an air parcel with z Zeng, . When ηηz ≠ 1, their spectrum evolution depends on the sign of 1 ‐ ηz. When ηz < 1, IR cools all ice crystals in the parcel; as a result, the relatively large ice crystals have a lower temperature than the small ones , which is studied next using two experiments: H07C and H11C.The bin model is used to simulate the REM of multiple ice crystals in an air parcel. Its results thus depend on the sign of 1 − e Figure , REM var3.2z = 7 km, where ηz = 0.86, T = ‐15.9°C, and p = 431 hPa. Since ηz < 1, the experiment simulates the effect of radiative cooling on ice crystal spectrum at z = 7 km or near top of the thick cloud A.Experiment H07C is designed to simulate REM at altitude The experiment uses 2,048 bins to represent differently sized ice crystals for a given crystal shape and orientation. Its initial relative humidity with respect to ice is 100%. All of the ice crystals are plate‐like and horizontally oriented. Their initial ice crystal spectrum is given byN(a) denotes the number of ice crystals with the semimajor axis length shorter than a, and c1 and c2 are two constants to be determined with initial IWC and ice crystal concentration (ICC).where −3 and an IWC of 1.5 × 10−3 g m−3, which is displayed by the thick line in Figure H07C takes the initial ice crystal spectrum with an ICC of 5.0 cma, and its vertical axis represents dM(ln a)/d ln a where M(ln a) denotes the mass of ice crystals with half‐crystal size shorter than a. The figure clearly shows that radiative cooling broadens the ice crystal spectrum and thus brings about large HOICs.Figure t, showing that the number concentration decreases with time. The figure also displays the time series of IWC, RHi, the average ice crystal sizeFigure ∫(a−a¯)2mdN(a)/∫mdN(a), and mean ice crystal fall speedice crystal spectrum width m and vf are the mass and fall speed of an ice crystal with the semimajor axis length a. As shown in Figure i decreases with time; in addition, the spectrum width, average ice crystal size, and mean ice crystal fall speed increase with time.where i or the water vapor pressure e of the parcel. Since ηz < 1, IR cools all ice crystals in the parcel and decreases the temperature of the relatively large ice crystals more than the temperature of the small ones .H11C and H07C broaden their spectrum with different time scale, because they have different temperature. Their spectrum broadening, as shown by the equation of diffusional ice crystal growth, depends on two factors: supersaturation and the saturation water vapor pressure. Since H11C and H07C have the same 3.4T and p on ice crystal sublimation  are carried out to obtain the time scale of REM at 5, 6, 8, 9, 10, 13, and 15 km altitude, respectively.The difference in the time scale between H07C and H11C comes from the effect of τ is defined asTo measure the crystal spectrum broadening in bulk, a time scale a¯0 at time t0 to a¯100 at time t100. Obviously, the time scale represents a period for ice crystals to double their average size.following Zeng , where tτ is computed, using the following parameters: t0 = 0, a¯0 = 9 μm, a¯100 = 100 μm, and t100 that is determined when the modeled average crystal size reaches 100 μm. Using Equation ηz = 0.86 in Table τ at nine altitudes is obtained and displayed by the red circles and black line in Figure The time scale τ depends on ηz, which is illustrated with two new experiments: H07C2 and H11C2. H07C2 and H11C2 take the same setup as H07C and H11C, respectively, except for ηz = 0.93. Their modeled time scales are displayed by two red circles in Figure ηz.On the other hand, the time scale ηz  can be estimated using the time scale at ηz = 0.86. Since τ is inversely proportional to 1 − ηz /(1 − ηz). The time scale at ηz = 0.93, for example, should be twice the time scale at ηz = 0.86, which is displayed by the blue line in Figure ηz with the time scale at ηz = 0.86.In fact, the time scale at the other magnitude of z Zeng, , the timτ and ηz, the spatial distribution of REM near cloud A (or C) can be inferred with the spatial distribution of ηz near the same cloud. On the other hand, although the thick clouds A and C have the similar distribution of ηz, they are different in REM (or GMI 166PD) because of their different altitudes (or T and p) , Figure ηz: ηz can be decomposed into two parts. That isAlthough the sign of 1 − (ηz)background represents ηz with no cloud and (ηz)cloud a perturbation of ηz imposed by clouds. In other words, the sign of 1 − ηz is determined by the factors of both (ηz)background and (ηz)cloud.where (ηz)cloud represents a perturbation of ηz imposed by clouds, (ηz)cloudis proportional to IWC and LWC approximately. When clouds are optically thin , (ηz)cloud ≈ 0 so thatSince (ηz)cloudhas so large an amplitude that ηz possesses a dipole structure .When clouds are thick , ηz in Equation ηz < 1 near cloud top and ηz > 1 near cloud bottom, no matter where they occur.Based on the decomposition of ηz with cloud height and latitude. Cloud B, for example, is at 7 km with 1 ‒ ηz > 0, whereas cloud D is at 12.5 km with 1 ‒ ηz < 0. In view of the opposite sign of 1 − ηz between clouds B and D, thick and thin clouds are divided into midlevel and high‐level clouds, yielding four types of clouds whose samples are denoted as clouds A–D in Table Thin clouds may change their sign of 1 ‒ 4.1(ηz)cloudhas a dipole structure so that ηz < 1 near cloud top and ηz > 1 near cloud bottom. The dipole structure impacts HOICs at cloud top and bottom oppositely, which is simulated by two experiments of HV07C and HV07W , respectively.In a thick cloud , ηz < 1  there. Their fate near cloud bottom is simulated by another experiment of HV07W.HV07C suggests that large HOICs form near cloud top due to radiative cooling. Once the large HOICs form, they fall through the cloud while a part of them becomes randomly oriented due to turbulence Klett, . They evηz = 1.14. Its initial ice crystals consist of two parts: the original small ice crystals in the air parcel and the large ones that fall in from cloud top. To be specific, its initial ice crystal spectrum is equal to that of HV07C plus the spectrum of HV07C at 4 h, which is shown by the thick lines in Figure HV07W takes the same setup as HV07C except for ηz = 1.14) increases their temperature more than others. Meanwhile, the small HOICs grow via vapor deposition, because the radiative warming increases their temperature less than others. In addition, the small vertically oriented ice crystals shrink first and then grow. As a result, REM decreases the preference of an ice crystal orientation near cloud bottom, approaching to no preference.The evolution of ice crystal spectrum in HV07W is displayed in Figure In summary, HV07C and HV07W predict that there are more HOICs near cloud top than near cloud bottom. This prediction on the vertical distribution of HOICs is supported by the lidar observations in the Arctic cloud suggest that there are HOICs near top of thick clouds. Since the GMI 166PD is sensitive to HOICs near cloud top or the upper portion of clouds  bring about a high 166PD of 5 K, whereas the high‐level clouds  bring about a relatively low 166PD. In other words, the higher the cloud top, the lower the 166PD. Such a difference in 166PD between the midlevel and high‐level thick clouds is caused by their difference in the REM time scale , and 166V Tb  in a range of 160 and 250 K background. Their value of ηz, or (ηz)background, is computed using Equation ηz < 1 below 10 km and ηz > 1 above 10 km in the tropics and middle latitudes, whereas ηz < 1 throughout the troposphere in the high latitudes during wintertime.Thin clouds  change (ηz)background, HV07C predicts that the ice crystals with horizontal orientation have more chance to grow than those with other orientation below 10 km. Another similar experiment, HV11W, is carried out to show no preference for horizontal ice crystal orientation above 10 km.Based on the vertical distribution of ηz = 1.14, T = ‐42.9°C, and p = 246 hPa. Thus, one‐half of its initial ice crystals are horizontally oriented and the other half are vertically oriented. It simulates how radiative warming impacts the thin clouds at 11 km . Its results show no obvious preference for crystal orientation (figure not shown), because the ratio of major to minor axis length is close to one when ice crystals are small  Since ηz usually decreases with latitude background < 1 throughout the troposphere, especially during wintertime , large ice crystals grow in expense of water vapor, whereas small ones shrink to supply water vapor, maintaining a quasi‐balance in water vapor between the large and small ice crystals. Once an upward motion occurs , it changes the quasi‐balance by providing additional water vapor for deposition. To be specific, the upward motion increases supersaturation so that large HOICs grow fast while the small ones need no sublimation to supply water vapor.This modeling result of large HOICs is consistent with the GMI observation: high 166PD over the cloud anvil near mesoscale convective systems (MCSs). Figure 5.2Ice crystal habit (or shape) varies with temperature and supersaturation  is computed with satellite‐retrieved cloud variables  near cloud top and warming (or ηz > 1) near bottom. Hence, the bin‐model simulations predict that HOICs occur near thick cloud top, which is consistent with the high frequency of positive GMI 166PD over thick clouds  are predicted by bin‐model simulations first and then traced in cloud observations. For a practical study, clouds are sorted into several groups according to ηz, T, and vertical velocity) will be incorporated into a cloud/radiation parameterization to better simulate clouds in weather and climate models.The general agreement between the bin‐model simulations and cloud observations suggests that REM does exist in many cirrus clouds. The common high GMI 166PD indicates that REM impacts cirrus clouds broadly. In the future, REM with its factors (e.g.,


Non-rainfall moisture  is an important driver of plant litter decomposition in grasslands, where it can contribute significantly to terrestrial carbon cycling. However, we still do not know whether microbial decomposers respond differently to NRM and rain, nor whether this response affects litter decomposition rates. To determine how local moisture regimes influence decomposer communities and their function, we examined fungal communities on standing grass litter at an NRM-dominated site and a rain-dominated site 75 km apart in the hyper-arid Namib Desert using a reciprocal transplant design. Dominant taxa at both sites consisted of both extremophilic and cosmopolitan species. Fungal communities differed between the two moisture regimes with environment having a considerably stronger effect on community composition than did stage of decomposition. Community composition was influenced by the availability of air-derived spores at each site and by specialization of fungi to their home environment; specifically, fungi from the cooler, moister NRM Site performed worse  when moved to the warmer, drier rain-dominated site while Rain Site fungi performed equally well in both environments. Our results contribute to growing literature demonstrating that as climate change alters the frequency, magnitude and type of moisture events in arid ecosystems, litter decomposition rates may be altered and constrained by the composition of existing decomposer communities. Understanding when and how microbial communities are driven by different climate variables and when compositional shifts have functional consequences will help us better predict how carbon cycling will change under changing climate conditions.Decomposition of organic matter is a major source of greenhouse gas emissions in terrestrial systems and plays an essential role in ecosystem carbon and nutrient cycling . Biotic One of the most important climatic factors determining microbial community structure and decomposition rates is moisture availability . While rThere are several reasons why microbial communities could differ in NRM- and rain-dominated environments. First, in many systems fog and dew occur more regularly than rainfall , so orgaWe provide the first assessment of how fungal communities are differentially influenced by NRM and rainfall. Specifically, we addressed the following research questions:(1) How does moisture regime (NRM- versus rain-dominated) drive fungal community composition?(2) Do changes in fungal community composition driven by moisture regime influence rates of litter decomposition?We hypothesized that differences in the air-derived fungal communities (fungi that colonized pre-sterilized tillers) as well as selection for different moisture regimes would lead to different fungal communities under the two regimes. We further hypothesized that some fungi would specialize in a particular environment. Finally, we hypothesized that these differences in community structure would affect decomposition rates, with fungi decomposing litter faster under their native moisture regime than in non-native conditions.1 maintained by the Gobabeb Research and Training Centre2. At the Rain Site, we used a HOBO data logger  and sensors to record temperature and relative humidity (S-THB-M002), rain (Davis S-RGD M002), and leaf wetness (S-LWA-M003). Measurements were recorded once per minute and converted into hourly averages. The weather station at the Rain Site failed two months before the end of the experiment, so we used data from a nearby weather station . An analysis comparing weather data from the two eastern stations (Dieprivier and the Rain Site) for ten months before the failure showed that the two sites had comparable weather regimes. Since solar radiation can accelerate litter decomposition by photodegradation  and has two very different moisture regimes in close proximity to one another: the western region is dominated by fog and dew and receives scant rainfall while the inland eastern areas receive more rainfall but very little non-rainfall moisture . Work waradation  and alteradation , we estiradation .To see whether fungal communities functioned similarly under NRM- versus rain-dominated conditions, we reciprocally transplanted litter between the two sites . HereaftStipagrostis sabulicola at the two sites. S. sabulicola is the dominant grass in the Namib Sand Sea and grows in hummocks on unstable dune faces . We confirmed this in the present study by visually examining tillers for evidence of sand blasting after collection.Despite the fact that this study was conducted in a sand dune system, sandblasting and aeolian-driven mass loss were not substantial mass loss drivers in this study. First, we used only coarse tillers, leaving no attached leafy material that would be shredded or easily removed, even during very high winds . SecondlWe used tillers at two different stages of decay to see whether moisture regime structured communities differently as tillers decomposed. Early-stage tillers were harvested from recently senesced stems that still had inflorescences attached (< 2 months post-senescence), were yellow, had no visible fungal growth, and had a visibly intact cuticle. Late-stage tillers were harvested from upright stems that had been standing for at least one year. These were characterized by coverings of light and dark-pigmented fungi and a cracked cuticle that was considerably more permeable to water . The priTo assess how colonization from air-derived fungi affected litter community assembly, we deployed “bait tillers,” which were autoclaved early-stage tillers collected from the NRM Site. We included bait tillers from only one site because space limitations in our litter racks prevented us from including bait tillers from both sites. Sterilization was confirmed by plating tillers on culture media. Autoclaving is an imperfect means of sterilization because it can alter associated chemistry but since our goal was to identify air-derived fungi, this deployment of standardized bait tillers allowed us to estimate the contribution of air-derived fungal sources  since the only fungi present on autoclaved tillers after one year would have been deposited from the air. Importantly, any confounding effects of autoclaving on litter properties would have been the same for litter deployed at both sites, allowing us to use a standardized litter type to compare air-derived fungal communities at the two sites.We measured C:N ratios in litter on a Thermo Finnigan Flash-1112EA microanalyzer . We used an ANKOM 200 Fiber Analyzer and Daisy Incubator  to measure acid detergent lignin, cellulose, and hemicellulose of live tillers and a subset of early- and late-stage tillers to determine initial litter chemistry at the start of the study. To determine whether mass loss was driven solely by differences in leaching from tillers at the two sites, we measured leaching on a subset of recently senesced tillers collected from the two sites that were identical to those used in our decomposition study. We sealed the cut ends of each tiller with glue, submerged them in ultrapure water at 4°C for 24 h, dried them, and weighed them to quantify mass loss due to leaching.PRJNA685174.Fungal communities were analyzed using molecular methods. We extracted DNA from tillers using a MoBio PowerSoil DNA extraction kit supplemented with 10 min of sonication prior to mechanical lysis. We sequenced the ITS region  using 250-bp paired-end sequencing on the MiSeq Illumina (V2) platform at Michigan State University’s Research Technology Support Facility Genomics Core. We used the same clustering and filtering pipeline used for Namib fungal (ITS) sequences described elsewhere . All seq3. Merged sequences were quality filtered to an expected error threshold of 1.0 fastq_filter . We also removed two OTUs that were identified as S. sabulicola, the host plant.Reads per sample ranged from 1,805 to 52,926 averaging 22,425. We used multiple extraction blanks to check for contamination, eventually removing three OTUs from the dataset. After OTU clustering, we removed any taxa that fit all three of the following criteria: it had more than 100 reads when summed across three blanks, had never been found in any previous sequencing datasets from working in the Namib Desert, and had never been cultured from the Namib in previous studies e.g., . This leS. sabulicola tillers in previous studies in the Namib, which had been identified with the longer ITS 1-4 region (∼600 bp)  .Saccharomyces cerevisiae as a standard to quantify ITS gene copies and normalized this to the dry mass of litter used for the extraction to produce a proxy of fungal biomass reported as log10 ITS copies gram–1.As a proxy of fungal biomass, we used quantitative PCR to count the number of ITS gene copies per sample and normalized it to the tiller’s dry biomass . While tt-test and lm functions and analyzed the reciprocal transplant experiment with Type III ANOVAs using the Anova function in the car package. PERMANOVAs and NMDS ordinations were generated using Bray-Curtis distances calculated using the distance function in the phyloseq package  and more frequent rain events than the NRM Site. The average time between wet events (either rain or NRM events) was 33 hours at the NRM Site and 90 hours at the Rain Site (p = 0.001). Dry periods lasting for more than 100 hours were not uncommon at the Rain Site  during the study period was 9% greater in the vicinity of the Rain Site than the NRM Site, though these were based on estimates from proxy sites and likely overestimate differences between our sites. Both sites had similar bidirectional NW and E wind regimes with seasonal variation in wind direction, suggesting similar source regions of air-derived fungi, though the Rain Site had a stronger southern component , Neostagonospora caricis (OTU8), and Phaeococcomyces mexicanus (OTU30)) together accounted for more than 53.0% of reads, but less than 1.4% at the Rain Site (Aureobasidium melanogenum (OTU3), Phaeococcomyces sp. (OTU17), Alternaria alternata (OTU1)) accounted for 50.8% of Rain Site reads but only 9.2% of NRM Site reads.Fungal communities on native tillers differed between the sites . Site exain Site . LikewisR2 = 0.23, P = 0.008)  , they al= 0.008) ; the 73 = 0.008) , suggest= 0.008) .Aureobasidium melanogenum (OTU3), unknown Pleosporales (OTU104), and Alternaria alternata (OTU1)) made up 31.0% of the Rain Site air-derived reads and 25.8% of the NRM Site air-derived reads. The top three air-derived taxa at the NRM Site  made up 43.7% of NRM Site air-derived reads and 10.2% of Rain Site air-derived reads  and Aureobasidium melanogenum (OTU3)] were highly abundant on both bait tillers and native tillers at both sites.The three most abundant air-derived taxa at the Rain Site . Early-stage tillers moved to the NRM Site had higher fungal biomass than those that remained at the Rain Site  and Aureobasidium melanogenum (OTU3) each represented over 10% of read abundances on native or bait tillers at both sites , Neophaeothecoidea spp. (OTU4), Phaeococcomyces mexicanus (OTU30), and Selenophoma linicola (OTU33). Despite their low abundance at the warmer, drier Rain Site, most of these taxa are closely related to organisms known for their desiccation and thermal tolerance. Neophaeothecoidea is a monotypic genus first isolated from protea plants in South Africa and, while its ecology has not yet been described in detail, it’s confamiliars in Teratosphaeriaceae occupy a wide range of habitats including highly acidic soils  and Phaeococcomyces species (OTU17). Pseudopithomyces is often found on dead plant litter and as a pathogen of some plants , is a congeneric of a fungus with a strong preference for the NRM Site (Phaeococcomyces mexicanus OTU30), suggesting that the traits responsible for thriving under the NRM-dominated versus rain-dominated environment may not be so clearly delineated along taxonomic lines.Two fungi showed much greater abundances at the Rain Site than the NRM Site: e plants  includine plants . The clo regions . InteresOverall, we did not find clear differences in desiccation tolerance or general stress tolerance among the fungi that preferred the NRM Site versus the Rain Site based on our literature searches. While culture-based follow-up studies may provide more information about the desiccation and thermal tolerances of these organisms, biotic interactions may also play a role in determining which taxa dominate at each site. For instance, while some NRM Site fungi may be fully capable of surviving the abiotic conditions of the warmer, drier Rain Site, they may simply be outcompeted by others with even slightly greater dessication tolerance. Similarly, Rain Site fungi appear fully capable of utilizing NRM when given the opportunity , but oveThe fact that both of these sites harbored diverse fungi associated with such wide-ranging niches was surprising, since we might have expected that only specialized fungi could tolerate the radiation and desiccation stressors of the hyperarid Namib Desert. Instead, several of the most abundant taxa were found worldwide in mesic and arid environments. This could be partially explained by considering the traits that support wide distributions. In bacteria, some of the traits that permit growth in an arid, sunny environment (such as pigmentation) correlate with large geographic ranges , since tBy using bait tillers, instead of direct air sampling, to assess the air-derived fungal community, we focused on those fungi that were capable of colonizing senesced grass tillers. These results show that the airborne and litter-associated fungal communities appear to interact with one another, representing a shared pool of dominant fungi. Although we were unable to determine whether air-derived communities at these two sites differed primarily due to differences in local plant diversity at the two sites, long-distance dispersal from wind, or other factors, we did find that the two sites contained a large pool of shared taxa. Cosmopolitan taxa (those that were present at both sites) make up the vast majority of reads on bait tillers at both locations, suggesting that similar air-derived species pools are available to colonize litter at both sites. Overall, litter-associated communities differed between the sites much more than air-derived communities did, suggesting that post-colonization processes play a role in structuring fungal communities differently at the two sites.Fungal communities on some transplanted tillers changed, as would be expected if some fungi performed better in one environment than the other. Fungal communities on early-stage tillers shifted while well-established late-stage tillers retained their initial communities . That we2 respiration  as the sole driver of fungal community differences that we observed, though it was likely the dominant factor. We collected standing dead litter from a sand dune system and suspended it aboveground so any differences in soil type would not be a factor. NRM duration differed by a factor of five to ten between the sites while minimum and maximum temperatures were far more comparable . The Raipiration  and littpiration , even wipiration  found thpiration , so we wpiration . Future S. sabulicola tillers at these same locations, found that the relationship between litter gravimetric moisture content and CO2–C flux did not differ between the two sites, demonstrating that fungi from both sites respire similarly under NRM. Our results showing that Rain Site fungi moved to the NRM Site decompose litter at the same rate as native NRM Site fungi would seem to corroborate this. Taken as a whole, this suggests that optimization for fog and dew by NRM specialists may not reflect any particular ability to utilize NRM so much as their inability to cope with the harsher  conditions associated with the Rain Site.Although we did not attempt to identify specific traits that drove differentiation in fungal communities at the two sites, our results suggest that the ability to tolerate the harsher conditions of the warmer, drier rain-dominated environment is an important driver of fungal differentiation across this gradient. The NRM-to-Rain transition may be difficult because the Rain Site is warmer, experiences less frequent wetting, greater solar irradiance, longer dry periods, and shorter NRM events  Table 1, conditiMicrobial biomass strongly regulates decomposition at regional scales and can predict how decomposition rates respond to changing climatic conditions . Fungal Plant litter decomposition is influenced by many factors, including photodegradation , litter The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/KJ and PJ conceived of the study, conducted fieldwork, and edited the manuscript. SE conducted data analysis and edited the manuscript. JL conducted lab work, data analysis, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.