The seasonal melting of ice entombed cryoconite holes on McMurdo Dry Valley glaciers provides oases for life in the harsh environmental conditions of the polar desert where surface air temperatures only occasionally exceed 0°C during the Austral summer. Here we follow temporal changes in cryoconite hole biogeochemistry on Canada Glacier from fully frozen conditions through the initial stages of spring thaw toward fully melted holes. The cryoconite holes had a mean isolation age from the glacial drainage system of 3.4 years, with an increasing mass of aqueous nutrients  with longer isolation age. During the initial melt there was a mean nine times enrichment in dissolved chloride relative to mean concentrations of the initial frozen holes indicative of an ionic pulse, with similar mean nine times enrichments in nitrite, ammonium, and dissolved organic matter. Nitrate was enriched twelve times and dissolved organic nitrogen six times, suggesting net nitrification, while lower enrichments for dissolved organic phosphorus and phosphate were consistent with net microbial phosphorus uptake. Rates of bacterial production were significantly elevated during the ionic pulse, likely due to the increased nutrient availability. There was no concomitant increase in photosynthesis rates, with a net depletion of dissolved inorganic carbon suggesting inorganic carbon limitation. Potential nitrogen fixation was detected in fully melted holes where it could be an important source of nitrogen to support microbial growth, but not during the ionic pulse where nitrogen availability was higher. This study demonstrates that ionic pulses significantly alter the timing and magnitude of microbial activity within entombed cryoconite holes, and adds credence to hypotheses that ionic enrichments during freeze-thaw can elevate rates of microbial growth and activity in other icy habitats, such as ice veins and subglacial regelation zones. The McMurdo Dry Valleys (MDV) are polar deserts comprising the largest ice-free expanse in Antarctica, and represent one of the coldest and driest ecosystems on Earth   have highly elevated ionic concentrations up to two orders of magnitude greater than in fully thawed holes, with large diurnal variations  in cryoconite holes through the start of the melt season on Canada Glacier in the MDV. This study will increase our understanding of cryoconite hole biogeochemistry, and help to define the role of cryoconite holes in the depauperate ecosystem of the MDV.T = 0, T + 3, T + 5, and T + 10 . Eight ice cores with no underlying cryoconite were also sampled on 25th November 2011. All cryoconite holes were completely frozen at T = 0, with increasing melt water at later time points. The same cryoconite holes were not sampled repeatedly; rather, eight different cryoconite holes were destructively sampled in the same local 10 m2 area of the glacier centered around the following coordinates: S 77°37.546′, E 162°57.620′. In all cases, samples were taken using a hand driven Kovacs ice corer. Prior to sampling, the width and length of the cryoconite holes  were measured using a ruler (±0.5 cm).The sample site was located on the surface of Canada Glacier, located within the 34 km long Taylor Valley of the MDV, Antarctica. Eight ice-lidded cryoconite holes were sampled at four different time points on 25th November, 28th November, 30th November, and 5th December 2011, hereafter termed After coring, ice cores and fully frozen cryoconite holes were transferred to large polyethylene bags for and transported to the field laboratory. In the laboratory, cores were melted in the laboratory and samples treated as below for water samples. In cryoconite holes where liquid water was present, the ice lid was removed and the water depth, sediment depth, and hole depth were measured with a ruler (±0.5 cm). Water was sampled using a 50 ml plastic syringe and transferred into 1L HDPE bottles for later ion and nutrient analysis and 65 ml glass BOD  bottles for dissolved inorganic carbon (DIC) and pH measurements later the same day. All HDPE bottles were rinsed three times with sample prior to filling. The BOD bottles were filled from the bottom using a length of Tygon® tubing attached to the syringe, and the bottle allowed to overflow 3× their volume prior to capping with no headspace. Cryoconite samples were collected with a stainless steel scoop into 50 ml Falcon tubes. Ice samples were collected in polyethylene bags, melted in the laboratory and the water treated as below for water samples.2 evolved using a portable PP systems EGM-4 infrared CO2 meter, with a coefficient of variation of 1%  and cations  were analyzed on a dual system Thermofisher Dionex 5000 capillary ion chromatograph with IonPac AS11-HC (0.4 × 250 mm) and CS16 (0.5 × 250 mm) columns at 30°C, using 32.2 mM KOH and 30 mM methanesulfonic acid (MSA) as eluents for anions and cations, respectively. Detection limits for Cl−, NO−3, and SO2−4 were 0.5, 0.6, and 0.2 μmoles L−1 respectively, with coefficients of variation of 0.8, 2.4, and 1.9% (n = 10). Detection limits for Na+, K+, Ca2+, Mg2+ were 2.7, 1.4, 1.0, and 0.1 μmoles L−1 respectively, with coefficients of variation of 1.8, 2.7, 1.9 and 1.9% (n = 10). Dissolved organic carbon (DOC) was analyzed on a Shimadzu TOC-V CSN analyzer. The detection limit was 10.1 μmoles L−1 with a coefficient of variation of 6.4% (n = 6). NO−2 and NH+4 were analyzed on a Bran + Luebbe Autoanalyzer 3, with detection limits of 0.09 and 0.4 μmoles L−1 respectively, and coefficients of variation of 1.5 and 9.2%, respectively. PO3−4, total dissolved phosphorus (TP(aq)) and total dissolved nitrogen (TN(aq)) were analyzed on a Lachat QuickChem 8500 Series 2 Flow Injector Analyzer using standard QuikChem® methods 31-115-01-1-I, 31-115-01-3-F and 31-107-04-3-A, respectively. Detection limits were 0.036, 1.4, and 1.0 μmoles L−1 for PO3−4, TP(aq) and TN(aq) respectively, with coefficients of variation of 0.5, 3.4, and 7.4% (n = 6). Dissolved organic nitrogen (DON) was defined as TN(aq) − [NH+4 + NO−2 + NO−3]. Dissolved organic phosphorus (DOP) was defined as TP(aq)-PO3−4.Dissolved anions  was analyzed using a Picarro L1120-i cavity ringdown spectrometer, with a precision of ±0.2‰. Inorganic carbon speciation in T + 10 cryoconite holes (the only time point where pH was reliably measured) was modeled using the computer program PHREEQ-C using its standard database.The δ−1 and 100 μg N g−1 respectively, with coefficients of variation for duplicate analyses of 9.5 and 5.3%. Exchangeable NH+4, NO−3, and NO−2 in wet cryoconite were extracted using a 2M KCl method  in dried cryoconite were analyzed using a three stage sequential digestion method using (1) 1 M MgCl2, (2) 0.1 M NaOH and (3) acid persulfate. Extracts were analyzed on a Shimadzu mini UV-vis spectrophotometer. The average coefficients of variation for duplicate cryoconite samples for the MgCl2, NaOH, and persulfate digestion steps were 10.1, 6.2, and 10.3%, respectively.Three cryoconite samples from each time point were analyzed for TOC, IC, and TN. All eight cryoconite samples from each time point were analyzed for solid phase nitrogen and phosphorus speciation. Dried cryoconite samples  were analyzed for total organic carbon (TOC) and total nitrogen (TN) on a Eurovector EA3000 Elemental Analyzer. Detection limits for TOC and TN were 100 μg C g−1 final concentration) and samples filtered onto black 0.22 μm polycarbonate filters (Millipore). Over 300 stained bacterial cells per slide were counted in duplicate using epifluorescence microscopy, and procedural blanks (sterilized deionized water) were subtracted from the sample counts. The same procedure was followed to enumerate photoautotrophic microbes using chlorophyll autofluorescence instead of staining the samples with DAPI   from biovolume.Three cryoconite samples from each time point were analyzed for total cell counts, autofluorescent counts, and biomass estimates. All eight samples at each time point were assayed for potential photosynthesis, bacterial production, and potential nitrogen fixation. Total and autofluorescent cell counts were enumerated by epifluorescent microscopy  was added. For each sample triplicate light, triplicate dark , and triplicate killed  vials were incubated for 1.5 h in an ice bath under a lamp. The average photosynthetically active radiation (PAR) at the surface of the ice bath measured using an Apogee PAR sensor and meter was 100 μmoles m−2 s−1 with a spatial variation of ±20 μmoles m−2 s−1. It should be noted that the actual PAR levels below ice lids in the cryoconite holes were not measured hence these rates of photosynthesis should be viewed as potential only. Vials were incubated for 1.5 h then removed from the ice bath and filtered through 0.45 μm cellulose nitrate filters. The pH of the filtrate was immediately raised to >pH 10 by the addition of pellets of NaOH prior to disposal. The filters were fumigated with 50% glutaraldehyde and 37% v/v HCl for >1 h, and the filters placed in scintillation vials. Vials were transported to the main laboratory (McMurdo), where 10 ml of Cytoscint ES scintillation cocktail was added and DPM values measured using a Beckman LS6000 scintillation counter, calibrated using external quench standards. Rates of photosynthesis (μg C g−1 h−1) were calculated following the procedures of Telling et al. , where 90 μl of 100% ice cold trichloroacetic acid was added to samples, and the tubes centrifuged at 16,000 g for 10 min. The pellet was washed sequentially with 5% trichloracetic acid and 80% ethanol and samples dried and weighed. Finally, 1 ml of Cytosint ES cocktail was added to samples, and DPM counted on a Beckman LS6000 scintillation counter calibrated using external quench standards. Bacterial production rates (μg C g−1 h−1) were calculated following the methods of Anesio et al.  between fully frozen cryoconite holes and surrounding ice cores as follows:Enrichment factors, where for example ion and nutrient concentrations in cryoconite holes are normalized to mean concentrations in the surrounding ice, can greatly facilitate the identification of biogeochemical sources and sinks , and mean Cice is the mean concentration in the ice cores.where T + 3, T + 5, T + 10) and fully frozen cryoconite holes (T = 0) to facilitate the identification of biogeochemical processes occurring during early melt freeze-thaw effects as follows (using the example for T + n):Similarly, we define enrichment factors between partially melted cryoconite holes .where 18O of the water as a more quantitative proxy than time for the percentage of ice melt within each individual hole, since the initial melt water should be enriched in the lighter 16O-H2O. 16O-H2O preferentially partitions into the water phase relative to the heavier 18O-H20  using Equation 3 from Fountain et al.  that the initial cryoconite water and additional annual ice melt have chloride concentrations equivalent to the mean chloride concentration of sediment free ice and, (c) that the ice lids contain no chloride  after normalizing ion and nutrient data to the surface area of the holes in order to negate the impact of concentration and dilution of nutrients by freeze-thaw.The isolation age of the cryoconite waters from the surrounding supraglacial water system was estimated using chloride mass balance, following the approach of Fountain et al. . Chloridn et al. , assuminT = 0 and T + 10 cryoconite holes with the measured nutrient demands of bacterial production and photosynthesis:Comparison of the rate of influx of nutrients from ice ablation into cryoconite holes with rates of microbial growth can give insight into potential microbial nutrient limitations  or TP(aq)] from ice ablation into the hole (μmoles m−2 h−1), based on the concentration of nutrients in the ice, Cice, and the ablation rate, Z , D is the estimated nutrient demand  from bacterial production or photosynthesis within the cryoconite hole (μmoles m−2 h−1), activity rate is the measured rate of bacterial production or photosynthesis in the cryconite (μmoles g−1 h−1), M is the dry mass of cryoconite within the hole , the Redfield coefficient, R, is 1 for C, 1/16 for N, and 1/106 for P, and A is the plan view area of the cryoconite hole (m2).where F is the nutrient flux [DOC, DIC, TN−1 (Table 6) to potential carbon fixed (μmoles C g−1) assuming a C:N molar ratio of 6.6:1 .Mean water depths in the holes increased with time to a maximum of 12.1 ± 4.9 cm at 2+ (20.7×), Mg2+ (8.2×), and SO2−4 (9.1×) were higher than those of Cl− , Na+ (2.0×), and K+ (2.6×). All dissolved nitrogen and phosphorus species  were depleted relative to Cl− with enrichment factors <2× (Table Ice core and aqueous geochemistry of cryoconite holes is summarized in Table  SO2−4 9.× were hi SO2−4 9.× were hi18O-H2O during the first melt (T + 3) were lower relative to frozen cryoconite holes at T = 0 (Table − concentration in early melt waters (T + 3) was enriched 9.3× over fully frozen cryoconite holes (T = 0), alongside >9× increases in mean NO−3, NO−2, Mg2+, and DOC concentrations , while the mean DIC was depleted at T + 3 (0.5×) relative to T = 0. A plot of DIC against DOC indicates two distinct groups of samples: those with a relatively constant DOC level but varying DIC concentration  and those with a relatively constant DIC concentration and higher and varying DOC concentrations  between δ18O-H2O and log enrichment factors for Cl−, SO2−4, NO−3, NO−2, TN, Mg2+, Ca2+, DON, DOP, and DOC and a less strong (p < 0.05) correlation between δ18O-H20 and the log enrichment factor for K+  or between δ18O-H20 and log enrichment factor for DIC   concentrations varied from 0.05 to 12.9 μmoles L−1, with a mean of 2.6 ± 4.7 μmoles L−1.pH was only measured in cryoconite holes at (aq) in ice cores was 4.5 ± 1.3 (Table (aq) of cryoconite holes were elevated relative to the surrounding ice, bracketing the Redfield ratio at 5.9–9.6 (Table p < 0.01) between δ18O-H2O and DOC:TP(aq) but not between δ18O-H2O vs. DOC:TN or δ18O-H20 vs. TN:TP(aq)  , the remainder contained <0.1 mg N g−1 (Table The mean organic and inorganic carbon concentrations of cryoconite were 0.9 ± 0.4 and 0.5 ± 0.3 mg C gKCl varied with time. The lowest mean concentrations of NH+4KCl were at T = 0 (85 ng N g−1), with a maximum at T + 3 (962 ng N g−1) and reduced concentrations through T + 5 to a mean of 316 ng N g−1 at T + 10 , which fell to a mean of 3.1 ng N g−1 at T + 10 , with low concentrations of PMgCl2 (mean of 0.5 μg P g−1) and PNaOH (mean of 10.3 μg P g−1)  was 10.6 ± 6.8%  relative to other time points (0.9–6.5 μg C g−1 wet sediment)  and T + 10  (Table T + 3 and T + 5 (Table (aq) was ≤9μmoles L−1, and TNKCl was ≤330 ng N g−1  correlation between bacterial production and potential photosynthesis . There was a significant correlation of bacterial production with δ18O-H2O  but not between potential photosynthesis and δ18O-H2O   from chloride mass balance was 3.4 ± 3.6 (1σ) years , Na+ , K+ , Mg2+ , Ca2+  and SO2−4  with increasing isolation age , DON , DIN , DOP  and PO3−4  with increasing isolation age of the holes. In contrast, there were no significant (p < 0.05) relationships between isolation age and DON:DIN , DOP:PO3−4 , PMgCl2 , PNaOH , Presidual  or NKCl  (not shown), bacterial production  or photosynthesis   of the cryoconite holes , and below 0°C for all but a few days of the entire melt season  and phosphorus species (DOP and PO3−4) relative to Cl− are consistent with the microbiological uptake of nutrients  and nutrients  with increasing isolation age of the holes . It is possible that trends could be masked by the high natural variation in microbial activity rates in individual samples  were lower than the mean reported for cryoconite holes from a range of MDV glaciers   are similar to those reported previously from Canada Glacier using the 14C method of 17–58 ng C g−1 h−1   were close to the detection limit (Table NaOH (mean of 10.3 ± 3.4 μg P g−1). The majority of phosphorus in cryoconite was in the form of Presidual (mean of 1455 ± 241 μg P g−1), of which approximately 40% may be in the form of organic phosphorus  from ice ablation alone are unlikely to be able to sustain the measured rates of microbial activity, even with efficient recycling between the heterotrophic and autotrophic communities Table . Particuit Table  consisteit Table  there weT = 0 and T + 10; Table 2H4 m−2 d−1) were similar to those measured in open cryoconite holes on a Greenland glacier of <4.2–16.3 μmoles C2H4 m−2 d−1  of cryoconite holes coincident with more negative δ18O-H2O  Cl− has the second largest enrichment factor after Mg2+  , while microbial activity could influence the concentrations of C, N, and P species.Relative to frozen cryoconite holes at 2O Table . The meating T +  of cryocse Table . For exating T +  of cryoc+4 (9.7×), and NO−2 (9.7×) were similar to Cl− (9.3×)  , NH) Figure , suggestigure −3 2.2×  increased rates of bacterial production  and DOP (4.4×) during the ionic pulse were lower than for DOC and dissolved nitrogen species, resulting in higher TOC:TP(aq) and TN:TP(aq)ratios  elevated during the ionic pulse  rather than enrichment in mean DIC concentrations during the ionic pulse event (Table 2(aq) concentrations of this study (T + 10 only) ranged from 0.05 to 12.9μmoles L−1, with a mean of 2.6 μmoles L−1. Freshwater phototrophs typically have a higher affinity for CO2 than HCO−3 , with a median of 13 μmoles L−1  in cryoconite holes at T+10. There is also a weak but significant correlation (p < 0.05) between DIC and rates of photosynthesis , driving DIC (and Ca2+) concentrations down  may have promoted higher rates of photosynthesis in an early more nutrient enriched melt water, both consuming DIC and producing alkalinity to further drive carbonate equilibria toward carbonate precipitation  of basal ice forms thin films of water, perhaps tens of microns thick (Raiswell et al., 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.


Objective: Applications for Abdominal Wall Vascularized Composite Allotransplantation may expand if a functional graft with decreased immunosuppressive requirements can be designed. We hypothesize that it is anatomically feasible to prepare a functional, innervated, and vascularized abdominal composite graft using a multilayered component separation technique. Including vascularized bone in the graft design may decrease the immunosuppressive requirements by inducing immunologic chimerism. Methods: Two cadaver torsos were used. Adipocutaneous flaps were elevated from the midaxillary lines, preserving deep inferior epigastric artery perforators. A 2-layered component separation through the external and internal oblique fasciae was carried out, exposing segmental intercostal thoracolumbar nerves. Superiorly directed muscle release over the subcostal margin provided for a 3-rib segment with attached rectus abdominis muscle. The remainder of the full-thickness allograft was harvested with its vasculature. Flap inset into the recipient cadaver abdomen, with osteosynthesis fixation between donor and recipient ribs, was achieved. Results: The harvested grafts had an average size of 845 ± 205 cm2 with a total procurement time of 110 minutes. On one cadaver, 4 thoracolumbar nerves were isolated bilaterally, while the other cadaver yielded 3 nerves. The nerves were transected with an average length of 5.7 ± 1.2 cm. The graft vasculature was transected with a length of 4.40 ± 0.10 cm. Conclusion: Using the principles of component separation technique, we demonstrated a novel approach to harvest and transfer a neurotized osteomyofasciocutaneous abdominal wall allotransplant as a multipedicled, single functional unit. Successful reports of upper extremity,3-Since the first description by Levi et al a decade ago, 2 groups (University of Miami and University of Bologna) have published their consecutive case series of abdominal wall transplantation. In total, they have published the results of 18 denervated AW-VCA in 17 patients.3We postulate the patient population that might benefit most from increased indications for abdominal wall transplantation without viscera or organ would be those people with massive “end-stage” hernias. Hernia disease burden in the United States is now epidemic and multifactorial in etiology. Patients with “end-stage” hernia disease, having total loss of domain with impaired biophysical and psychosocial function, represent a population with few viable options for the repair of the hernia and reconstruction of the abdominal wall. Because abdominal wall reconstruction is morbid, frequent, and often multirecurrent at initial presentation, there may finally be good reason and indication—in select patients—to consider a functional AW-VCA with immunosuppression for the reconstruction of massive composite defects.Expanding the indications for AW-VCA evokes the ethical questions of subjecting a patient to a lifetime of immunosuppression. To justify such a risk, the AW-VCA procedure must first improve the biomechanical function of the graft and also minimize the immunosuppressive needs. Abdominal wall surgeons have learned that bridged mesh repairs are less durable than primary closure with functional reconstruction of the anterior fascia. Primary closure is most commonly achieved through component separation technique . We note the rectus abdominis muscle flap is not simply vascularized but, more importantly, also remains innervated when returned to its midline position of function with physiologic tension. In the same sense, it stands to reason that any AW-VCA graft should also remain innervated after transplantation to improve its function. As studies look to improve the practicality of nerve transfers in other VCA models, the same must be continued for AW-VCA.11-The minimization of immunosuppression and induction of immune tolerance reported in VCA has made large strides in recent years. Directly transplanting hematopoietic stem cells has shown to decrease the immunosuppressive burden due to its chimeric effects on the recipient.12With the end-stage hernia patient in mind, drawing upon our large experience with component separation techniques, and improvement in VCA transplant biology, we now hypothesize that procurement of an innervated abdominal wall is technically feasible for allotransplantation. Our approach combines the concepts of functional hernia repair with the notion of innervated abdominal wall allotransplantation through preparation of the graft using a stepwise “multi-layered” component separation technique that carefully identifies individual segmental intercostal nerves beneath the internal oblique muscle. Secondarily, we hypothesize that an allograft can be designed with an osseous component to utilize the aforementioned immune-modulating chimerism effect.Two fresh cadaver torsos were latex injected and obtained from the Maryland State Anatomy Board on June 6, 2013. The operative plan was for the procurement of an osteomyofasiocutaneous abdominal wall allotransplant, which could be neurotized and inset as a single multipedicled functional graft. To this effect, a hexagonal skin incisional was planned see . IncisioSuperiorly directed muscle release was carried up and over the subcostal margin, similar to the component separation modification used in the repair of subcostal or epigastric hernias; this maneuver revealed the attachments of rectus abdominis to the lowermost ribs see . Using aThe allograft harvest was continued by carrying the dissection through to the peritoneum cavity beginning at the costal margin with full-thickness incisions in the abdominal wall between bony landmarks such as anterior superior iliac spine and symphysis see  and then2. The length of abdominal wall procurement was 110 minutes. As the skin flaps were elevated laterally to medially, at least 2 latex-filled perforators from the underlying deep inferior epigastric pedicle were easily identified on both cadaver hemiabdomens.The average size of the hexagonal abdominal wall graft harvested was 845 ± 205 cmThe 2-layered component separation technique readily allowed for the rapid isolation of the segmental intercostal thoracolumbar nerves. On 1 cadaver, 4 nerves were easily dissected on each side while the other cadaver yielded 3 nerves. The average length of the skeletonized thoracolumbar nerves was 5.7 ± 1.2 cm. We expect that with continued careful dissection each spinal level's intercostal contribution could be identified and that longer lengths of donor nerve could be obtained.When completing the graft harvest, 6-cm segments of the 3 inferior most vertebrochondral ribs were removed with the rectus muscle attached using a Stryker reciprocating power saw and osteotome. The deep inferior epigastric vessels were transected at their attachment to the external iliac vessels at a length of 4.40 ± 0.10 cm. Upon flap transfer, nerve coaptation was easily simulated between donor and recipient intercostal levels, and that osteosynthesis with internal fixation provided for stable bone to bone contact.If tissue coverage itself, without motor function, is the end point for a large abdominal wall composite defect such as a hernia, then there are an abundance of techniques already described . However, if the goal is restoration of a functional abdominal wall with a durable result, then an AW-VCA with motor function is theoretically a better option in the end-stage hernia patient who has already failed multiple previous attempts at reconstruction. A functional graft in essence means a neurotized and vascularized composite graft with the myofascial component being under proper physiologic tension. Such a functional graft would enable the abdominal wall to contribute to respiration, coughing, micturition, flexion movement, and core stability while at the same time preventing herniation of abdominal contents.-Current abdominal wall transplantations serve only as a denervated, poorly functional mechanical retainer of the abdominal contents.3In a cadaveric study, Ramirez et alThe first difference between techniques is that component separation, when used to repair abdominal wall defects with native tissue, is started by elevating broad skin flaps through midline laparotomy, progressing in a medial to lateral direction to gain access to the lateral abdominal wall. Conversely, the goal in allotransplantation is to preserve the midline structures and overlying blood supply to the skin. Thus, skin flaps are actually based medially on the deep inferior epigastric perforators and elevated off the underlying abdominal wall from a lateral to medial direction instead. Once the semilunar line is encountered, care is taken to preserve the forthcoming perforators, as they will provide the superficial blood supply to the skin (Huger Zone 1).18Second, instead of just preforming a single layer of separation, allowing access into the avascular plane between the external and internal oblique muscles, a “multilayered” separation is necessary. Continued separation of the internal oblique and transversus abdominis muscle exposes the thoracolumbar neurovascular bundles. The thoracolumbar nerves, T7 to L4, provide the motor innervation to the abdomen necessary for a functional graft. The separation for the second layer was initiated at a point 2-cm lateral to the semilunar line by carefully dissecting through the internal oblique musculature until the plane between the 2 muscles was identified. This location was chosen to avoid damaging the thoracolumbar nerves as they penetrate through the semilunar line.In our cadaveric study, we demonstrated that it is feasible to identify and to skeletonize adequate length of the thoracolumbar nerves. Through this novel approach, the thoracolumbar nerves could be easily isolated, harvested, and used for coaptation with the similarly prepared recipient thoracolumbar nerves. The longer the segment of nerve that can be harvested from a donor, the easier it will be to perform a neurorrhaphy with the corresponding recipient nerve, especially if the donor has considerable abdominal domain loss. Successful neurotization and implantation of an innervated graft would yield the possibility of a functional abdominal wall.,Another novel concept proposed by our cadaveric study is incorporating bone as part of the allograft. Immunosuppression regimens used in the abdominal wall combined with visceral or solid organ transplants result in an acceptably low rate of graft rejection for both components of the allograft, but the use of these regimens in an otherwise healthy individual with end-stage hernia poses significant ethical considerations. The potential advantages of including vascularized bone marrow are providing a more stable fixation of the allograft, setting the graft under physiologic tension, and reducing immunosuppressive requirements by inducing chimerism.14An alternative strategy for reducing the antigenicity of the flap itself would be to limit the amount of skin transplanted. Skin presents the highest concentration of antigens, naturally lending itself to higher rates of rejection. We suggest that de-epithelializing the graft could decrease the immunosuppressive requirements necessary. The wound could be regrafted with epidermal autografts or split-thickness skin autograft. In addition, we further imagine that an entirely skinless flap could be harvested from a donor, further decreasing the immunosuppressive requirements. Such a recipient patient could be covered with tissue expanded flaps, tissue rearrangement using thigh flaps, or simply with skin advancement flaps in the recipient. Cosmetically, this concept may be appealing as it provides for local tissue match with respect to surrounding skin quality, color, and tone.To address the possibility of insufficient vascular supply within the distal graft, the graft could be supercharged with a superior vascular anastomosis based on the lowermost subcostal/intercostal vessels, the superior epigastric system, and/or the superficial inferior epigastric system in addition to the primary vascular pedicle from the deep inferior epigastric vessels bilaterally. It is important to include the deep inferior epigastric system in any graft combination because it is the dominant blood supply to the rectus abdominis muscle. Even though the rectus abdominis muscle can survive with only the superior epigastric system, the perforators that supply the overlying skin, specifically the Huger zone 1 perforators, are primarily supplied by the deep inferior epigastric system.19It should be noted that incisional hernias are one of the most common types of hernias experienced by patients. It would prove counterproductive and be seen as a failure if a patient's hernia was repaired with an AW-VCA only to have them return with incisional hernia complications, especially after possibly exposing them to an immunosuppressive regiment. However, we argue that having an incisional hernia is more manageable than having total loss of the abdominal domain. Our group has shown that using biological mesh as a primary reinforcement of suture lines reduces the incidence of incisional hernias and also reduces postoperative wound infection rates in immunosuppressed posttransplant patients.,Expanding the indications for AW-VCA to include the end-stage hernia patient is technically possible. Similar to the challenges previously faced with lower extremity VCAs, much work remains to be done to refine the AW-VCA technique and identify the most appropriate patients for this procedure.21Vascularized composite allotransplantation is an established and rapidly expanding field that promotes the idea of quality of life in patients with severe pathology such as recalcitrant facial deformity and severe hand dysfunction. While abdominal wall transplantation is already a reality, we believe the provision of functional innervation may expand the indication to those patients suffering from debilitating end-stage hernia disease.Demonstrated here is a novel approach to the abdominal wall—using principles of component separation technique—to easily dissect the crucial segmental intercostal innervation within the deep lateral abdominal wall. This technique is feasible in a cadaver model indicating the possibility of osteomyofasciocutaneous free flap for allotransplantation. Furthermore, we illustrate the ability to include bone with orthopedic osteosynthesis for the purpose of inducing marrow-derived chimeric tolerance.


In vitro, signaling through CCL27-CCR10 mediates activation of p-Akt, and subsequently induces proliferation and invasive responses. Cell proliferation and invasion promoted by CCL27 were blocked by inhibition of p-Akt or CCR10. In vivo, down-regulation of CCR10 significantly impairs growth of glioma. Clinically, High CCR10 expression in GBM correlated with p-Akt, shorter overall survival and progression-free survival (P < 0.05). Together, these findings suggest that elevated CCR10 is a critical molecular event associated with gliomagenesis.Human gliomas are characterized by their invasion of normal brain structures irrespective of their grade of malignancy. Tumor cell invasion share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptor CCR10 is highly expressed in human glioblastoma compared with control brain tissue.  Glioblastoma is the most malignant primary brain tumor in human with poor survival despite multimodality treatment -3. In thChemokine receptors are cytokine receptors found on the surface of certain cells that interact with a type of cytokine called a chemokine. There have been 19 distinct chemokine receptors in mammals. Each has a 7-transmembrane structure and couples to G-protein for signal transduction within a cell, making them members of a large protein family of G protein-coupled receptors. Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux in intracellular calcium (Ca2+) ions . This causes cell responses, involved in leukocyte migration, angiogenesis, hematopoiesis and tumor developments. In addiCCR10 gene. Its ligands are CCL27 and CCL28. CCR10 is normally expressed by melanocytes, plasma cells and skin-homing T cells. Melanoma cell transduction of CCR10 significantly increases the development of lymph node metastasis in mice after inoculation in the skin. Besides, CCR10-CCL27 interactions also play a key role in T cell homing in inflamed skin and melanoma directing metastasis. However, the role of CCR10 in glioma was not well known. In this study, we report that CCR10 is highly expressed in human glioblastoma and correlated with shorter overall survival and progression-free survival. Signaling through CCL27-CCR10 mediates activation of p-Akt, and subsequently induces proliferation and invasive responses. These results suggest that CCR10 high expressed in glioma is essential for tumor proliferation, invasion and progression.So far, various chemokine receptors are expressed on plenty of cancers, including glioma, 17, 18.Chemokine receptors are well known for tumor proliferation and invasion. Here we use GBM gene expression profile from TCGA compared chemokine receptors with normal brain tissue. There are five chemokine receptors overexpressed in GBM, including CXCR4, CXCR7, CCR5, CCR7 and CCR10 Figure . We focuin vitro.To assess effects of CCL27 on glioma cells, we stimulated U87 and LN229 cells with recombinant human chemokine CCL27 under serum deprivation conditions and examed cell proliferation by cell count and MTT assay. Cell proliferation was increased by stimulation with CCL27 in a dose-dependent manner Figure . MoreoveAfter engagement with appropriate ligands, chemokine receptors trigger a complex cascade of intracellular signaling events that activate downstreem pathway. To identify the downstream pathways that may contribute to CCL27-CCR10 signaling, we performed gene expression profiling to compare the CCL27 high/low and CCR10 high/low GBM samples. Analyses of biological pathways using gene-set enrichment analysis (GSEA)  revealedin vitro.After administration of CCL27 to glioma cells, we observed a strong increase in p-Akt. The expression of p-Akt was nearly completely inhibited by selective PI3K inhibitors LY294002 Figure . To knocin vitro, we evaluated the contribution of CCL27/CCR10 signaling on tumor growth in vivo by using experimental intracranial models. Since CCR10 shRNA was more effective in LN229 than U87, we chose LN229 cell for the in vivo models. When the mice intracranially transplanted with LN229 that stably express luciferase and CCR10 shRNA, CCR10 shRNA resistant or control vector, bioluminescence imaging was done for the whole body. CCR10 shRNA treated LN229 cells displayed a marked reduction of the tumor , phosphoinositide 3-kinase (PI3K), STAT3, and NF-kB. However, little is known about downstream signaling pathways regulated by CCL27-CCR10 interaction in glioma. GSEA is a computational method developed at the Broad Institute of MIT and Harvard that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states. After c subtype . Thus, CIn summary, we show here that CCR10 is expressed in glioma and found that CCR10 expression correlates with poor survival of GBM patients. We propose the linkage between CCR10 and p-Akt, both of which play critical roles in carcinogenesis, has advanced our knowledge of how CCR10-overexpressing cancer cells mediated the glioma malignant.2 atmosphere. Cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.Human SNB19, U251, U87, T98, LN229 and LN308 glioblastoma cells were obtained from the China Academia Science Cell Repository, Shanghai, China. The cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Gibco), and were incubated at 37°C in a 5% CO80 paraffin-embedded gliomas specimens with clinical data were collected from Department of Neurosurgery, Huashan Hospital, Fudan University, including 10 grade II tumors, 10 grade III tumors and 60 grade IV tumors according to WHO classification (2007). 8 control brain tissues were obtained from the patients with severe traumatic brain injury (TBI) who needed post-trauma surgery. This study was approved by The Local Ethical Review Board of the Huashan hospital, Fudan University and written informed consent was obtained from all patients.CCR10 siRNA and CCR10 shRNA were designed by BLOCK-iT™ RNAi Designer, invitrogen, USA. siRNA_1: Sense: GCUGGAUACUGCCGAUCUA; Antisense: UAGAUCGGCAGUAUCCAGC. siRNA_2: Sense: GCCUCAAUCCCGUUCUCUA; Antisense: UAGAGAACGGGAUUGAGGC. shRNA_91_top Insert Sequence: CACCGCTTTGCTACAAGGCCGATGTCGA AACATCGGCCTTGTAGCAAAGC; shRNA_91_bottom Insert Sequence: AAAAGCTTTGCTACAAGGCCGATGTTT CGACATCGGCCTTGTAGCAAAGC. Cells were transfected with CCR10 siRNA or scrambled oligonucleotides and Lipofectamine 2000 (Invitrogen) for vitro study. Cells were transfected with CCR10 shRNA or control vector and Lipofectamine 2000 (Invitrogen) for vivo study.Recombinant human chemokine CCL27 were from Sino Biological Inc, added to cell cultures at a final concentration of 0.01 to 0.1μg/ml. PI3K inhibitor LY294002  was dissolved in DMSO at a stock concentration of 10 mM and added to cell cultures at a final concentration of 30 μM.Following the transfection of LN229 and U87, cell count and MTT assay was used. Cells were counted using the Coulter Counter (Beckman Coulter). MTT assay was done as previous study , 21. Eac2). Briefly, the Matrigel was allowed to rehydrate for 2 h at 37C by adding warm, serum-free DMEM. The wells of the lower chamber were filled with medium containing 5% fetal bovine serum. U87 and LN229 Cells (5 × 104) were seeded in the upper compartment (6.25-mm membrane size) in serum-free medium. Exogenous CCL27 (0.1μg/ml) were added in the upper chamber. After 24h incubation, non-invading cells were removed from the top well with a cotton swab, and bottom cells were fixed with 3% paraformaldehyde, stained with 0.1% crystal violet, and photographed in 9 independent 10× fields for 3 wells. Fold invasion was calculated relative to blank control. Data represents mean ± standard error (SE) of 3 independent experiments.Invasion was measured using 24-well BioCoat cell culture inserts (BD Biosciences) with an 8-μm-porosity polyethylene terephthalate membrane coated with Matrigel Basement Membrane Matrix  and luciferase lentivirus were injected into the intracranial of 5-week-old BALB/c-nu mice. At day http://tcga-data.nci.nih.gov/tcga/homepage.htm. Rembrandt data was downloaded from the following portal: https://caintegrator.nci.nih.gov/rembrandt/home.do. To quantify the visual pattern, Wilcoxon rank-sum test is used to measure the significance of differential gene expression between normal brain tissue and GBM in TCGA and Unpaired t test in Rembrandt. Overall survival curves were plotted according to the Kaplan–Meier method, with the log-rank test applied for comparison. All differences were considered statistically significant at the level of P < 0.05. Statistics were performed using the SPSS Graduate Pack 11.0 statistical software .The TCGA mRNA expression microarray data (AgilentG4502A_07_2 array N=483) and metadata including survival information for GBM patients were downloaded from the following portal:


Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu), mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP), and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP). Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.Miniature inverted-repeat transposable elements (MITEs) are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE  in trans by their autonomous element(s). It has been unknown how MITEs amplify themselves to high copy numbers in the genome. Our results demonstrate that the rice active MITE mPing is mobilized in the embryo by the developmental stage-specific up-regulation of an autonomous element, Ping, and thereby successfully amplifies itself to a high copy number in the genome. The short-term expression of Ping is thought to be a strategy of the mPing family for amplifying mPing by escaping the silencing mechanism of the host genome.Transposable elements are major components of eukaryotic genomes, comprising a large portion of the genome in some species. Miniature inverted-repeat transposable elements (MITEs), which belong to the class II DNA transposable elements, are abundant in gene-rich regions, and their copy numbers are very high; therefore, they have been considered to contribute to genome evolution. Because MITEs are short and have no coding capacity, they cannot transpose their positions without the aid of transposase, provided  Zea mays) and barley (Hordeum vulgare) genomes are composed of TEs Oryza sativa) and Arabidopsis , respectively Transposable elements (TEs) are DNA sequences that are capable of jumping from one genomic locus to another and make up a large fraction of eukaryotic genomes. More than 80% of the maize  that transpose through a copy-and-paste mechanism via an RNA intermediate, and class II elements (transposons) that transpose through a cut-and-paste mechanism without undergoing an RNA intermediate. Class I elements easily attain tens of thousands of copies, whereas the majority of class II elements cannot amplify themselves to 50 copies at most. Unlike other class II elements, miniature inverted-repeat transposable elements (MITEs) have the capacity to amplify themselves to high copy numbers (hundreds or thousands) Miniature Ping (mPing) is the first active MITE identified in the rice genome mPing exceptionally remains at a low level in most rice cultivars: indica and tropical japonica cultivars have fewer than 10 copies, and temperate japonica cultivars including Nipponbare have approximately 50 copies mPing is suppressed in most rice cultivars, but, like other TEs, mPing is activated by exposure to various stress conditions such as gamma-ray irradiation mPing transposition mPing is actively transposing without such stresses in the temperate japonica rice strain EG4 (cultivar Gimbozu) under natural growth conditions, and its copy number is approximately 1000 copies mPing has overcome the silencing mechanism or established a novel strategy for its amplification in the EG4 genome. In this sense, mPing in EG4 is an appropriate material to study the amplification of MITEs in plant genomes.Ping and its distantly related element Pong, which both belong to the PIF/Harbinger superfamily, provide two proteins required for mPing transposition. Both Ping and Pong have two open reading frames (ORFs), ORF1 and ORF2 Ping and PongPIF/Harbinger superfamily suggested that the Myb-like DNA-binding protein directly binds to the subterminal regions of the transposon in order to recruit the transposase Ping or Pong or both elements are necessary for mPing transposition The autonomous element mPing is actively transposing in the embryo of EG4 during the period from the regionalization of shoot apical meristem (SAM) and radicle to the formation of the first leaf primordium  with the aid of developmental stage-specific expression of Ping. Our results provide important evidence for the amplification mechanism not only of mPing but also of other MITEs.In this study, we demonstrate that mPing might overcome such a silencing mechanism in EG4. To confirm this hypothesis, we developed two F1 populations from reciprocal crosses between the mPing-active strain EG4 and the mPing-inactive cultivar Nipponbare, and investigated the transposition activity of mPing by transposon display (TD) analysis. Success of reciprocal crosses was confirmed by PCR analysis using locus-specific primers  for the mPing transposition is present in both male and female gametes of EG4.Plants have acquired the silencing mechanism of TEs in germ cells. In Arabidopsis, for example, TEs are activated specifically in the vegetative nucleus of the pollen, and siRNAs from the activated TEs accumulate in the sperm cells  primers . One of re plant . Furtheranalysis . We deterogenies . This coulations . MoreovemPing using genomic DNA samples extracted from endosperm, radicle, and leaf blades of eight progenies (S1) derived from a single parental EG4 plant (S0), and investigated the mPing transposition during ontogeny of rice plants  arises during the vegetative and reproductive phases.We performed TD analysis of e plants . One of e groups : (1) endanalysis . NumbersmPing scarcely transposes during the period from the onset of gametogenesis to the early stage (until 3 DAP) of embryogenesis. Among the 228 de novo insertions, 116 and 17 were shoot-specific and leaf-specific insertions, respectively  to the formation of the 3rd leaf primordia (at 8 DAP). Of the 133 shoot- and leaf-specific insertions, 108 were of the 1st leaf blade  derived from the main culm and the primary tiller of a single selfed parent (S1) to investigate whether the de novo insertions detected in ontogenical analysis are inheritable . In contrast, 85% (11 of 13) of the shoot-specific insertions that were detected in the S1 plants also appeared in the S2 progenies. This value (85%) is consistent with the estimated number of inheritable de novo insertions in our previous report de novo insertions that arose in the 3 to 5 DAP embryo were successfully inherited to the next generation.In order for eritable . Both ramPing insertions  in the EG4 genome mPing excisions in a small EG4 population using locus-specific primer pairs mPing excision with locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We randomly chose 48 markers for this study  analysis to compare the expression level of pponbare . Since Eone copy , the difat 3 DAP . The raty of EG4 . Since mPing by in situ hybridization using Ping-specific probes. The probe positions were indicated in Ping transcripts were detected in all tissues, viz. embryo, endosperm, and ovary wall, in both EG4 and Nipponbare , whereas Nipponbare has only one (Ping-N)  in the first intronic region of Ping-ORF1 was detected between EG4 and Nipponbare  . When wepponbare . Ping-N mPing activity Ping and the copy number of Ping and mPing in 93 AG strains, and evaluated the effect of C-type Ping on the mPing activity. These 93 AG strains were divided into three groups according to the SNP-type of the Ping allele  and two mPing-inactive strains (A105 and G190) during embryogenesis (from 1 to 6 DAP). A119 and A123 have six and ten copies of C-type Ping, respectively, and both A105 and G190 have one copy of T-type Ping  described in the In addition to EG4, several Aikoku and Gimbozu landraces (hereafter AG strains) are known to exhibit high g allele : strainsype Ping . Expressnd 3 DAP . This inmPing, which is the only active MITE identified in rice, as a material and analyzed the timing of mPing transposition in the mPing-active strain EG4. Consequently, we successfully found one mechanism of the mPing amplification; mPing most actively transposes during the period from the regionalization of the SAM and radicle to the formation of the first leaf primordium (3 to 5 DAP) by the developmental stage-specific up-regulation of the autonomous element Ping.Our final goal was to elucidate how MITEs attain their high copy numbers in the genome. To this end, we chose LORE1a in Lotus japonicus is activated in plants regenerated from de-differentiated cells and transposes in male germ cells by the pollen grain-specific LORE1a transcription, resulting in the asymmetric transposition of LORE1a in the reciprocal crosses between the active and non-active lines Tag1 in Arabidopsis shows germinal transposition activity in both male and female germ cells. Consequently, the reciprocal crosses show symmetric transposition of Tag1mPing transposition activity, which may suggest that mPing in EG4 transposes in both male and female germ cells. However, we obtained only a few de novo endosperm-specific and radicle/shoot-specific insertions in the ontogenical analysis, although we detected a number of de novo shoot-specific and radicle-specific insertions. We therefore concluded that most mPing transposes not in germ cells but in somatic cells after pollination. Somatic transposition that occurs at the late stage of plant development often produces spotted and striped segments in tissues, such as maize seed coat variegation caused by Mutator excision from the bz2 gene nDart1excision from the OsClpP5 gene mPing elements transposed in somatic cells of the embryo during the period from 3 to 5 DAP. Being a class II TE that transposes by a cut-and-paste mechanism, mPing is expected to be eliminated from genomic DNA with a certain frequency. However, a previous report demonstrated that the mPing excision sites would be repaired by utilizing a copy of mPing from either the sister chromatid or from the homologous chromosome mPing excision site cannot be repaired if mPing transposes in germ cells, which are haploid. It is therefore considered that the somatic transposition of mPing is an important factor for mPing amplification in the genome.The transpositions of TEs are categorized into germinal and somatic types according to the type of cells where the transposition takes place. Ping and Pong mediate mPing transposition in the rice genome. Many japonica cultivars, including EG4 and Nipponbare, have both Ping and Pong. This study demonstrated that Ping plays a predominant role in mPing transposition in EG4. However, a heterologous expression assay using Arabidopsis and yeast showed that Pong had a higher catalytic capacity for mPing transposition than PingmPing transposition was observed under stress conditions in several rice cultivars harboring only PongPong through the development of rice plants, indicating that Pong would be epigenetically silenced at the transcriptional level in EG4. In contrast, Ping constitutively expressed in all organs including the SAM and embryo. Nevertheless, mPing could be transposing most actively in the embryo during the period from 3 to 5 DAP. Since the stage-specific up-regulation of Ping was observed during the period of mPing transposition, we hypothesized that the expression level of Ping needed to exceed a certain threshold of mPing transposition.The autonomous elements mPing-active strains  showed higher expression of Ping with a peak around 3 DAP than the mPing-inactive strains . Although further experiments are needed to elucidate the mechanism of developmental stage-specific up-regulation of Ping expression, we propose two hypotheses: (1) position- and dosage-effect, and (2) effect of SNP. The details of the hypotheses are as follows.All Tam3 copies residing in the Antirrhinum majus genome show different transposition activities from each other Tag1 increases in proportion to its copy number mPing-inactive strains Nipponbare, A105, and G190 have only one Ping at the same locus, whereas the mPing-active strains EG4, A119, and A123 have respectively seven, six, and ten copies of Ping at different loci except for the Ping-1 locus. Furthermore, the expression pattern of Ping showed slight variation among the mPing-active strains harboring only C-type Ping. These results suggest that the developmental stage-specific up-regulation of Ping expression is probably regulated by the position-effect and/or the dosage-effect.Chromosomal position and copy number of TE often affect the transposition activity. The former is known as ‘position effect’ and the latter as ‘dosage effect’. Eight independent SLC22A4 affects transcriptional efficiency in vitro, owing to an allelic difference in affinity to the transcriptional factor RUNX1 Ping-ORF1, and Ping elements in the AG strains were categorized into either T-type or C-type Ping according to the SNP-type. Since all strains that showed a peak in the expression analysis had only C-type Ping, the intronic SNP might influence the developmental stage-specific up-regulation of Ping expression. T-type Ping was present in 14 AG strains as one copy, and its chromosomal location did not differ among strains. In contrast, the copy number of C-type Ping varied from one to ten, and their chromosomal locations, except for Ping-1, differed from each other. These results indicate that T-type Ping has lost its activity, whereas C-type Ping may be still active in the rice genome. Furthermore, we found that the copy number of mPing was significantly larger in strains harboring C-type Ping than in strains harboring T-type Ping. This strongly supports that C-type SNPs in the intronic region of Ping contribute to the amplification of mPing, presumably by the developmental stage-specific up-regulation of Ping expression.Intronic SNPs are known to cause drastic effects on gene expression. In humans, an intronic SNP in mPing is transiently induced by various stresses mPing is suppressed by the silencing mechanisms in many cultivars. Thus, mPing must overcome the silencing mechanism in order to maintain the transposition activity under natural growth conditions. Our results revealed that mPing in EG4 was mobilized by the sufficient supply of Ping transcripts produced only during the period of mPing transposition. This stage-specific activation is thought to be a strategy of the mPing family to amplify mPing by escaping from the silencing mechanism of the host genome. Since no active MITEs other than mPing so far have been identified, it is very difficult to elucidate if the other MITEs also attain their high copy numbers in the same way as mPing amplifies. Given that the other active MITEs are identified, however, our study will help to understand their amplification mechanisms. Our previous study documented the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible mPing, other MITEs also contribute to gene and genome evolution via providing new promoter regulatory sequences, transcriptional termination elements, and new alternative exons Since the transposition of TEs often damages the host genome, TEs with high transposition activity are targeted by the silencing mechanisms. Nevertheless, MITEs amplify to very high copy numbers not only in plant genomes but also in animal genomes. Very little is known about how MITEs attain their high copy numbers by escaping the silencing mechanism. The transposition of 1) derived from a single parental plant (S0) were grown in a greenhouse, and all S2 seeds were harvested. For S1 plants, each seed was cut into two halves, and the half including the embryo was germinated and the other was sampled. After germination, the radicle and the 1st, 2nd, 3rd, 4th, and 5th leaf blades were sampled. The second leaf was collected from S0 and S2 plants. For estimation of Ping and mPing copy numbers, eight bulked plants were sampled. For RNA extraction, ovaries before pollination and ovaries at 1, 2, 3, 4, 5, and 6 DAP were collected. All samples were immediately frozen in liquid nitrogen and stored at −80°C until use.EG4 (cultivar Gimbozu), Nipponbare, and 94 Aikoku/Gimbozu landraces were used in this study . Aikoku/DNA extraction and transposon display was performed following a published protocol mPing-flanking fragments excised from transposon display gels and primer design were performed following a published protocol mPing insertion sites were forecasted by a BLAST search in the Rice Annotation Project Database  mPing flanking sequences as queries. To prepare enough templates for PCR, whole genome amplification was performed using an illustra GenomiPhi V2 Kit . mPing excision was detected by PCR with mPing-sequence characterized amplified region (SCAR) markers Ping-N, -1, -2, -3, -4, -5, -6, and -7, eight Ping-SCAR markers were used. The genomic locations of the Ping insertion sites were referred from a previous report Ping-1 allele, PCR was performed in 10-µl reaction volumes containing 10 ng of template DNA, 0.2 U of KOD FX Neo (Toyobo), 1×PCR buffer for KOD FX Neo (Toyobo), and 0.2 µM of each primer , 5% DMSO, and 0.25 µM of each primer  and digested using RNase-free DNase (TaKaRa). First strand cDNA was synthesized using a Transcriptor first strand cDNA synthesis kit (Roche). For reverse transcription PCR, PCR was performed in 10 µl reaction volumes containing cDNA generated from 4 ng total RNA, 0.2 U of KOD FX Neo (Toyobo), 1×PCR buffer for KOD FX Neo (Toyobo), and 0.5 µM of each primer. PCR conditions were as follows: 94°C for 3 min; 35 or 45 cycles of 98°C for 10 s, 60°C for 10 s, and 68°C for 10 s. Relative quantification of Ping-ORF1 (1091 bp) and Ping-ORF2 (1368 bp) were cloned into pBlueScript SK+ (Stratagene) and sequenced. For digoxigenin-labeled antisense/sense RNA probe synthesis, in vitro transcription was performed using T7 RNA polymerase and T3 RNA polymerase. In situ hybridization and immunological detection with alkaline phosphatase were performed according to Kouchi and Hata Plant samples were fixed with 4% (w/v) paraformaldehyde and 1% Triton X in 0.1M sodium phosphate buffer for 48 h at 4°C. They were then dehydrated in a graded ethanol series, substituted with 1-butanol, and embedded in Paraplast Plus. The samples were sectioned at 8-µm thickness using a rotary microtome. Fragments of Afa I restriction site, one mutation was introduced into the reverse primer. The 5-µl PCR products were mixed with 5 µl restriction mixture containing 1 U Afa I (TaKaRa), 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 0.5 mM Dithiothreitol, 66 mM K-acetate, and 0.01% (w/v) bovine serum albumin. After 16 h incubation at 37°C, DNA gel electrophoresis was performed. PCR products (502 bp) including +1261T SNP were not digested, whereas PCR products including +1261C SNP were digested into two fragments (352 bp and 150 bp).PCR was performed in 10-µl reaction volumes containing 10 ng of template of DNA, 5 µl of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 µM of each primer. PCR conditions were as follows: 94°C for 3 min; 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s; and 72°C for 1 min. PCR primers used in this study are listed in Ping by Southern blot analysis, genomic DNA samples were digested with Eco RI restriction enzyme. These samples were loaded onto an agarose gel, separated by electrophoresis, blotted onto a nylon membrane, and probed with the Ping fragment. The mPing copy number was determined by real-time quantitative PCR as described previously To determine the copy number of Figure S1Ping with locus-specific markers. The maker names and cross combinations are indicated at the top of the profiles. (B) Locus-specific PCR analysis of mPing in Nipponbare genome. The genomic location of the mPing insertions and cross combinations are indicated at the top of the profiles. G and F indicate parental EG4 plants and the F1 plants, respectively. Lane M: DNA size marker , Lane N: Nipponbare. Black and white arrowheads show the bands indicating the presence and absence of Ping/mPing, respectively.Verification of the reciprocal crosses between EG4 and Nipponbare. (A) Locus-specific PCR analysis of (TIF)Click here for additional data file.Figure S2de novo mPing insertions in F1 progenies. Ten representative results are shown. The genomic locations of the mPing insertions and cross combinations are indicated at the top of the profiles. G and F indicate parental EG4 and the F1 plants, respectively. Lane M: DNA size marker , Lane N: Nipponbare. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively.Locus-specific PCR analysis of (TIF)Click here for additional data file.Figure S3mPing transposition in EG4 by transposon display. Eight progenies (S1) were derived from a single parental EG4 plant (S0). The 2nd leaf blade of the S0 plant and the endosperm, radicle, and leaf blades of each S1 plant were sampled and subjected to transposon display. White, red, and green arrowheads indicate shoot-, radicle-, and leaf-specific insertions, respectively. The rice plant has alternate distichous leaves; therefore, we analyzed the insertion in both [n+1]th and [n+2]th leaves to confirm whether the insertion detected in [n]th leaf is leaf-specific or shoot-specific. But we did not investigate the specificity of the insertions detected in the 4th and 5th leaves using their upper leaves. For this reason, we did not categorize such insertions and marked with the gray arrowhead. E: endosperm; R: radicle; L1–L5: 1st to 5th leaf. For progeny 2–8, samples are applied in the same order as for progeny 1.Ontogenical analysis of (TIF)Click here for additional data file.Figure S4de novo mPing insertions in various tissues of a single EG4 plant. Eight representative results are shown. The genomic locations of the mPing insertions and insertion types are indicated at the top of the profiles. Lane M: DNA size marker , Lane N: Nipponbare, Lane E: endosperm, Lane R: radicle, Lane L1–L5: 1st to 5th leaf. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively.Locus-specific PCR analysis of (TIF)Click here for additional data file.Figure S5mPing transposition. If mPing transposes in the period indicated by the red bar, the schematic banding patterns indicated by the arrows will be obtained. E: endosperm, R: radicle, L1–L5: 1st to 5th leaf blade, DAP: days after pollination, SAM: shoot apical meristem.Schematic representation of the relationship between banding patterns obtained in transposon display and the timing of (TIF)Click here for additional data file.Figure S6de novo mPing insertions in EG4. S2 plants derived from the main culm and the primary tiller of a single S1 plant were assayed. The shoot-specific insertion in the S1 plant (white arrowhead) was inherited by S2 plants, whereas the radicle-specific insertion (red arrowhead) was not. E: endosperm; R: radicle; L1–L5: 1st to 5th leaf.Inheritance of (TIF)Click here for additional data file.Figure S7mPing excisions in EG4. mPing excisions were detected by locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We analyzed 48 loci. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively. Figures indicate  shoot-specific excisions,  leaf-specific excisions, and (E) radicle-specific excision. G: EG4 (S0 plant); N: Nipponbare; E: endosperm; R: radicle; L1–L5: 1st to 5th leaf.(TIF)Click here for additional data file.Figure S8Ping-ORF2 spatial expression patterns by in situ hybridization analysis. Longitudinal sections through the ovary 3 days after pollination of (A) EG4 and  Nipponbare were hybridized with  antisense or (C) sense RNA probes.Detection of (TIF)Click here for additional data file.Table S1De novo mPing insertion sites detected in F1 progenies.(XLSX)Click here for additional data file.Table S2De novo mPing insertion sites detected in various tissues of a single EG4 plant.(XLSX)Click here for additional data file.Table S3mPing-SCAR markers used in this study.(XLSX)Click here for additional data file.Table S4Plant materials used in this study.(XLSX)Click here for additional data file.Table S5Ping-SCAR markers used in this study.(XLSX)Click here for additional data file.Table S6Primer pairs used in this study.(XLSX)Click here for additional data file.


Hypothesis is a tightrope swinging between reality and a supposed truth.Valdeci Nogueira, Brazilian philosopher and writer.We are aware that all information published by each issue of this journal needs the squeezeand sweat of thousands of people. On one end of the rope, researchers and higher educationinstitutions work hard to produce science to the highest degree of excellence. On the otherend, reviewers silently filter manuscripts, thus improving the quality and accuracy ofinformation that has been approved. Science is tough, as it tests and audits one's dreams.But both ends of the rope face the same direction.th anniversary, we had to thank publicly everyone who contributed tothe significant growth of this journal, especially in the last few years: researchers,reviewers and higher education institutions which believed in building a Brazilianscientific journal to excellence. The idea is not to stretch the rope, so as to createcompetition; but strengthen it while acknowledging all contributors' competence andthanking them for their support which is key to the development of this journal. And we didso at the 10th Congress of Associação Brasileira de Ortodontia (BrazilianAssociation of Orthodontics- ABOR) held in Florianópolis / Santa Catarina, Brazil. At theevent, we disclosed information about the last three years of the journal, reporting theparticipation of more than 70 higher education institutions - from Brazil, United States,Canada, Italy, England, Colombia, Syria, Turkey and Pakistan - which believed in DentalPress Journal of Orthodontics (DPJO)'s credibility. We assessed the journal's data andreported the top ten most active institutions, all of which were Brazilian: University ofSão Paulo (USP)/ Bauru School of Dentistry (FOB-USP), Federal University of Rio de Janeiro(UFRJ), State University of São Paulo (UNESP)/ Araraquara, State University of Maringá(UEM), State University of Southwestern Bahia (UESB), Federal University of Bahia (UFBA),Ingá College (UNINGÁ), University of São Paulo (USP)/ School of Dentistry, State Universityof Rio de Janeiro (UERJ), Federal University of Juiz de Fora (UFJF). All of them areacknowledged for their hard work in graduate and/or undergraduate education, and trust inour journal's earnest and quality. On our 19We also reported the most productive authors published in DPJO. A total of 758 researchersfrom dozens of countries, among which some of the most important researchers of worldwideOrthodontics contributed effusively: Guilherme Janson (FOB-USP), Antonio Carlos Ruellas(UFRJ), Karina Salvatore de Freitas (Ingá College), Ary dos Santos-Pinto(UNESP/Araraquara), Carlos Nelson Elias (Military Institute of Engineering - IME), DanielaGarib (FOB and HRAC-USP), José Fernando Castanha Henriques (FOB-USP), Rodrigo HermontCançado (Ingá College), Matheus Pithon (UESB), Eduardo Franzotti Sant'Anna (UFRJ), OrlandoTanaka (PUC/PR), Leopoldino Capelozza Filho  and Mauricio deAlmeida Cardoso (USC). Researchers of inestimable value, pulling the rope towards DPJO andBrazilian Orthodontics growth.If on one hand, we congratulate those who produced so much science, we certainly have tohonor the other end of the rope: professionals who worked hard to filter the quality ofinformation, so as to render it more reliable for practical applicability in society. In2015, more than 180 reviewers, from different continents, contributed to such anindispensable task. Between 2013 and 2015, the most active were: Marcio Rodrigues deAlmeida (UNOPAR), Daniela Feu Laignier (UVV-ES), Alexandre Simões da Motta (UFF), EmanuelBraga Rego (UFBA), Alex Pozzobon Pereira (UFMA), Soraya Coelho Leal (UNB), Daltro Ritter(UFSC), Andre Wilson Machado (UFBA), Hugo Caracas (Brasília/DF) and Arno Locks (UFSC).Orthodontics thanks and feels proud for all the work you have done.If the rope has two ends, someone needs to balance the force originating in each one ofthem. All scientific journals have this delicate role played by the editors. At present,DPJO counts on the contribution of ten editors. They are scientists coming from differentparts of Brazil as well as overseas, all dully acknowledged worldwide. We thank profuselyall the work carried out by Professors Telma Martins de Araujo (UFBA), Luciane de Menezes(PUC-RS), Daniela Garib (USP), Fernanda Angelieri (UMESP) and Flavia Artese (UERJ), as wellas Professors Matheus Pithon (UESB), Ildeu Andrade (PUC-MG), Leandro Silva Marques (UFVJM)and Carlos Flores-Mir (University of Alberta - Canada).If hypothesis is a tightrope swinging between reality and a supposed truth, as described bythe epigraph, we must as well be proud of our rope. Dental Press Journal of Orthodonticshas operated for nearly 20 years as a strong rope supporting the bridge of scientificknowledge. The colossal extent of this bridge might bother and put us down, showing howsmall we are. Perhaps we are not even a rope, maybe a cord in the navel of science, only.We thus take this opportunity to realize how much we still have to grow.David Normando - Editor-in-chief (davidnormando@hotmail.com)


Auxiliary partial heterotopic liver transplantation (APHLT) with portal vein arterialization is a valuable procedure to be considered in the treatment of patients with acute liver failure and metabolic liver diseases. The aim of this study was to develop a new rat model of APHLT with liver dual arterial blood supply (LDABS). A total of 20 rats were used. The donor liver was resected, and the celiac trunk was reserved. Left and medial hepatic lobes accounting for 70% of the liver mass were removed en bloc and the suprahepatic caval vein was ligated simultaneously. Thus, 30% of the donor liver was obtained as the graft. Sleeve anastomosis of the graft portal vein and splenic artery were performed after narrowing the portal vein lumen through suturing. The right kidney of the recipient was removed, and sleeve anastomosis was performed between the celiac trunk of the graft and the right renal artery of the recipient. In addition, end-to-end anastomosis was performed between the infrahepatic caval vein of the graft and the right renal vein of the recipient. Following the reperfusion of the graft, the blood flow of the arterialized portal vein was controlled within the physiological range through suturing and narrowing under monitoring with an ultrasonic flowmeter. The bile duct of the graft was implanted into the duodenum of the recipient through an internal stent catheter. A 70% section of the native liver  was resected using bloodless hepatectomy. The mean operative duration was 154.5±16.4 min, and the warm and cold ischemia times of the graft were 8.1±1.1 min and 64.5±6.6 min, respectively. The blood flow of the arterialized portal vein to the graft was 1.8±0.3 ml/min/g liver weight. The success rate of model establishment  was 70% (7/10). Following successful model establishment, all rats survived 7 days post-surgery . The graft was found to be soft in texture and bright red in color following exploratory laparotomy. In conclusion, a new rat model of APHLT with LDABS without stent for vascular reconstruction was developed. This is a feasible and reliable rat model for liver transplantation study. Auxiliary liver transplantation (ALT) is usually used for the treatment of acute liver failure and inherited metabolic liver disease ,2. The aet al developed a rat model of APHLT with PVA  presents significant advantages, such as that the hilum and portal vein of the native liver are untouched, the surgical trauma of more extensive liver dissection of the native liver is avoided, and adequate portal venous blood flow of the graft liver is guaranteed. For these reasons, several clinical studies using APHLT with PVA have been conducted in recent years –6; howevwith PVA . In thisFor this purpose, a new rat model of APHLT with PVA was developed, based on a modification of the Schleimer model, using the splenic artery of the graft liver for arterialization of the portal vein instead of a stent.The experimental protocol was approved by the Laboratory Animal Ethics Committee of the Inner Mongolia Medical University .Male Sprague-Dawley (SD) rats, each weighing 300–350 g, were used in this study . The body weight of the donors and recipients were comparable. All procedures were performed by two persons using a clean but not sterile technique with a Leica M525 F20 surgical microscope . Anesthesia was induced by inhalation of 3–4% isoflurane, and maintained by inhalation of 1–2% isoflurane. All rats were given oxygen at a dose of 0.3–0.5 l/min.After anesthesia was induced, a crucial incision was made in the abdomen of the rat. The ligament surrounding the liver was cut off with an electric coagulation scalpel, the left phrenic vein and right suprarenal vein were ligated and divided, and the infrahepatic caval vein was isolated and injected with 2 ml low molecular weight heparin sodium salt , allowing systemic heparinization of the rats. The hepatoduodenal ligament was dissected, and the common bile duct was isolated. The lower segment of the common bile duct was incised, and a stent catheter appropriately 1 cm in length  was implanted, followed by double ligature with 8-0 nylon sutures. The main portal vein was isolated, and the pyloric vein was ligated and divided. The celiac trunk and its branches were isolated, and the left gastric artery, splenic artery and gastroduodenal artery were ligated with 8-0 nylon sutures and divided. The splenic artery stump was retained at 5 mm in length at least for the subsequent sleeve anastomosis, and the common hepatic artery and proper hepatic artery were reserved .The donor liver was perfused through the portal vein. After clamping the origin of the celiac trunk, the distal portal vein was clamped and incised, and a perfusion catheter was implanted. Perfusion was performed with 30 ml lactated Ringer’s solution containing low molecular weight heparin sodium (12.5 IU/ml), using the gravity perfusion method at a rate of 45 drops/min at 0–4°C. The infrahepatic caval vein was rapidly incised to allow outflow of the blood and perfusate. The celiac trunk was transected at its origin and rinsed with lactated Ringer’s solution containing low molecular weight heparin sodium (12.5 IU/ml). During the perfusion, the donor liver was persistently rinsed with physiological saline at 0–4°C until the liver became khaki in color. The donor liver was completely resected and stored in lactated Ringer’s solution at 0–4°C.The hepatic pedicle and hepatic vein of the left and medial liver lobes were ligated with 6-0 nylon sutures, and the left and medial hepatic lobes accounting for 70% of the liver mass were removed en bloc and the suprahepatic caval vein was ligated simultaneously. Thus, 30% of the donor liver was obtained as the graft. All lumens were rinsed with low molecular weight heparin sodium salt (50 IU/ml) and trimmed to facilitate the anastomosis. A portion of the lumen of the portal vein was sutured and closed with 10-0 nylon sutures to make the internal diameter of the portal vein lumen and external diameter of splenic artery comparable, and then sleeve anastomosis between the splenic artery and portal vein was performed .Following the successful induction of anesthesia, a median incision was made into the abdomen. The right renal artery and vein were isolated, and the origin of the right renal artery and the site for the right renal vein entry into the infrahepatic caval vein were clamped. The right kidney was then resected. The graft was implanted into the right renal fossa, and an inverted continuous all-layer suture was performed between the infrahepatic caval vein of the graft and the right renal vein of the recipient. Sleeve anastomosis was performed between the celiac trunk of the graft and the right renal artery of the recipient. Following successful anastomosis, the right renal venous clamp and right renal arterial clamp were removed successively to allow reperfusion; rapid recovery of the color of the graft, pulsation in the proper hepatic artery and celiac trunk, and cystic dilatation of the proximal portal vein were observed. The graft was warmed by rinsing with warm physiological saline, and bile was found to be discharged from the biliary stent catheter. Then, a stent catheter was implanted into the duodenum of the recipient, followed by purse-string suturing with 8-0 nylon sutures. To avoid dislocation and blood flow disturbance of the graft, the donor’s ligated suprahepatic caval vein was sutured and fixed with the right lateral abdominal wall .Following completion of the vascular reconstruction of the graft, the blood flow of the arterialized portal vein was measured using an ultrasonic flowmeter , and controlled within the physiological range (from 0.8±0.2 to 2.4±0.8 ml/min/g liver weight)  was resected en bloc by bloodless hepatectomy. The Glisson’s system of two hepatic lobes was ligated with 6-0 nylon sutures and then divided. The proximal portal vein was punctured and slowly infused with 3 ml lactated Ringer’s solution . Then, the hepatic vein was wrapped and ligated with 6-0 nylon sutures, and the left and medial hepatic lobes were resected successively in a clockwise direction. The abdominal cavity was infused with 5 ml warm lactated Ringer’s solution, and two-layer interrupted suture of the abdominal wall was performed with 3-0 silk sutures.All rats were fasted for 24 h prior to the transplantation, and were given free access to water. During the transplantation, a heating blanket was applied to the recipient rats to keep the body temperature at 36°C. The recipient rats were awake within 3 min after the transplantation, and then were able to perform turn-over activities. A high post-operative body temperature was maintained, and all rats had free access to food and water. In addition, 2 ml low molecular weight heparin sodium salt (50 IU/ml) was injected subcutaneously daily following the surgery. Antibiotics and analgesics were not administered generally.In total, 20 rats were used to establish the rat models of APHLT (10 as donors and 10 as recipients). The mean operative duration was 154.5±16.4 min, and the warm and cold ischemia times of the graft were 8.1±1.1 min and 64.5±6.6 min, respectively. The blood flow of the arterialized portal vein to the graft was 1.8±0.3 ml/min/g liver weight, as measured by the ultrasonic flowmeter, which was within the physiological range.The blood flow at all anastomotic stomas was smooth following transplantation. Waking with a post-surgical survival of >24 h was defined as successful model establishment. In this study, the success rate of model establishment was 70% (7/10), and three rats died due to long operative duration, large trauma and bleeding of the anastomotic stoma in the 24 h after surgery. The rat had free access to food and water following the surgery. All rats were able to perform normal activities, had glossy hair, and were alert. All rats that were successfully modeled survived 7 days post-surgery . The graft was found to be soft in texture and bright red in color following exploratory laparotomy.In the present study, a new rat model of APHLT with PVA was developed, characterized by: i) the graft splenic artery is reserved on the celiac trunk from the donor; ii) the graft portal vein is directly sleeve anastomosed to the graft splenic artery for arterialization of the portal vein; iii) the graft celiac trunk is sleeve anastomosed with the right kidney artery of the recipient; iv) the graft infrahepatic caval vein is end-to-end anastomosed with the right renal vein of the recipient. Furthermore, the study introduces a new term instead of PVA to describe this vascular reconstruction method; since the blood supplies of the graft liver in both the hepatic artery and portal vein are from same source , the term is liver dual arterial blood supply (LDABS). LDABS differs from arterioportal fistula , partialet al  during the preparation of the donor liver, the isolation is difficult to perform due to the thin diameter of the celiac trunk and its branches, and the isolation may stimulate spasm of the proper hepatic artery, leading to the possible effect of transient ischemia on the donor liver; ii) the large differentiation in the diameter between the portal vein and splenic artery (~3-fold) leads to difficulties in anastomosis. In this study, a portion of the portal venous lumen was sutured and closed and then sleeve anastomosed with the splenic artery. The blood flow of the portal vein was 1.8±0.3 ml/min/g liver weight, as measured by the ultrasonic flowmeter, which was within the physiological range. However, the anastomosis is challenging to perform, and the operator should have a high-level microvascular anastomosis technique; iii) the hepatic artery and portal vein of the graft receive the same arterial blood supply from the recipient’ right renal artery via the celiac trunk of the graft, which avoids vascular reconstruction using the recipient’s abdominal aorta, and causes minor trauma to the recipient; and iv) end-to-end anastomosis of the infrahepatic caval vein of the graft and the recipient’s right renal vein is used to build the blood reflux pathway. This method may avoid the blockage of the recipient’s infrahepatic caval vein during the anastomosis, and reduce the effect on the recipient’s systemic state; however, this pathway is likely to develop venous return obstruction owing to vascular distortion. Therefore, the length of the infrahepatic caval vein of the graft and the recipient’s right renal vein should be shortened as much as possible so as to widen the venous outflow of the graft. Exploratory laparotomy one week after the surgery revealed that the graft was soft in texture and bright red in color.et al in pigs (In future, if the vascular reconstruction developed in this model is considered for application to large animals or even to human subjects, the iliac artery could be used instead of the renal artery, such as described by Fernández-Rodríguez  in pigs .This study has a limitation, which is that no histological analysis or serum tests of liver enzyme activity were performed to evaluate the functioning of the graft liver. Future studies to perform these are planned.In conclusion, a new rat model of APHLT with LDABS without stenting for vascular reconstruction was developed. This is a feasible and reliable rat model for liver transplantation study.


We aimed to evaluate the association between biomarkers of hemodynamic stress and aortic stiffness assessed at a chronic stage after ST-segment elevation myocardial infarction (STEMI).Fifty-four patients four months after acute STEMI were enrolled in this cross-sectional, single-center study. N-terminal pro B-type natriuretic peptide (NT-proBNP), mid-regional pro-A-type natriuretic peptide (MR-proANP) and mid-regional pro-adrenomedullin (MR-proADM) levels were measured by established assays. Aortic stiffness was assessed by the measurement of pulse wave velocity using velocity-encoding, phase-contrast cardiovascular magnetic resonance imaging.NT-proBNP, MR-proANP and MR-proADM concentrations were all correlated with aortic stiffness in univariate analysis . In multiple linear regression analysis, NT-proBNP  and MR-proADM  levels were associated with increased aortic stiffness independently of age, blood pressure and renal function. In receiver operating characteristic analysis, NT-proBNP was the strongest predictor for high aortic stiffness .At a chronic stage after STEMI, concentrations of biomarkers for hemodynamic stress, especially NT-proBNP, are positively correlated with aortic stiffness. These biomarkers might also be useful as predictors of high aortic stiffness post-STEMI.Austrian Society of Cardiology.


I am honored to assume the responsibility of the Editor-in-Chief (EIC) of the Journal of Epidemiology. For the last 6 years, 2002-2007, the editorial committee has been successfully managed under the strong leadership of the former EIC, Dr. Yosikazu Nakamura. During his term as the EIC, the journal has achieved the fundamentals required to become an international scientific journal. The journal successfully obtained an impact factor in 2005 in the first attempt and scored 1.240 in 2006. The journal has been issued bimonthly since 2000 and distributes over 30 articles annually. Its successful management is reflected in the extraordinarily speedy responses after article submission: the first reviewers' comments reach authors within an average of 18 days since the receipt of an article.The journal will continue to serve the research community across a broad range of exciting developments in epidemiologic research. First, I ask all members of the Japan Epidemiological Association to continually contribute to the journal by submitting significant research experiments. However, this alone is insufficient to further establish our excellence as an international scientific journal. Although based in Japan, the journal welcomes contributions from all over the world, especially from Asian countries. The importance and significance of the work it publishes is reflected in its international subscriptions and readership outside Japan. In order to maintain this high standard, the editorial committee will continue to make every effort to improve the quality of the journal.My own field of study is cancer epidemiology. However, I can assure you that the journal is not about to become a journal of cancer. We shall publish the best of all epidemiologic researches.Tomotaka Sobue, MD, MPHEditor-in-ChiefThe Journal of EpidemiologyCancer Information Services and Surveillance DivisionCenter for Cancer Control and Information ServicesNational Cancer Center5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, JapanPhone: +81-3-3542-2511, ext: 3424Fax: +81-3-3546-0630E-mail: edit-je@cied2.res.ncc.go.jp


Thus, new kinds of dual-band or wide-band dual-mode SIW filters with a fixed or an independently reconfigurable low-frequency band have been directly achieved. In order to experimentally verify the proposed design method, four two-cavity dual-band SIW filters, which have different numbers of perturbation via-holes in each cavity, and a two-cavity dual-band SIW filter, which includes four via-holes and eight reconfigurable states in each cavity, are designed and experimentally assessed. The measured results indicate that the available frequency-ratio range from 1 to 1.3 can be realized by using four two-cavity dual-band SIW filters. The center frequency of the first band can be tuned from 4.61 GHz to 5.24 GHz, whereas the center frequency of the second one is fixed at around 6.18 GHz for the two-cavity dual-band SIW filter with four reconfigurable states via-holes. All the simulated and measured results show an acceptable agreement with the predicted data.A novel perturbation approach using additional metalized via-holes for implementation of the dual-band or wide-band dual-mode substrate integrated waveguide (SIW) filters is proposed in this paper. The independent perturbation on the first resonant mode TE Concurrent multi-band or real-time tunable band radio-frequency/microwave/terahertz transceivers provide effective and productive solutions to overcome the efficient-utilization problem in spectrum congested wireless communications in the real world. As a key building block of modern wireless transceiver systems, concurrent multi-band or real-time tunable band filters require multi-function integrated features to satisfy the increasing demands for the spectrum in future emerging applications in telecommunication systems and Internet of Things. Recently, a number of multi-band and tunable/reconfigurable filters have been reported in the literatures including coplanar waveguide filters34678910There are only two categories of SIW-based tunable/reconfigurable filters: 1) the electrically tunable filters6791011679101167891011101 mode) is studied, the effects of additional metalized holes on two modes (the TE101 and TE102 modes) are considered in this paper and the two modes are utilized for generating dual pass bands. Moreover, the length of slots in dual-band SIW filter101, but cannot change the second resonant mode TE102 in the proposed SIW filters. The use of the proposed perturbation holes at the center of each cavity significantly increases the flexibility of developing dual-band or wide-band tunable band SIW filters. Based on the outlined description of the operating principle and mechanism, two kinds of dual-band SIW filters, i.e., four two-cavity SIW filters with fixed dual bands and a two-cavity reconfigurable dual-band SIW filter, have been designed, fabricated, and experimentally evaluated. The measured results show that four two-cavity dual-band SIW filters with a frequency-ratio ranging from 1 (wide-band case) to 1.3  can be realized. On the other hand, the center frequency of the first band can be tuned from 4.61 GHz to 5.24 GHz, but the center frequency of the second band will be unaltered and fixed at around 6.18 GHz for the two-cavity dual-band SIW filter with four reconfigurable via-holes. All the simulated and measured results are in good agreement with the predicted data, thus verifying the proposed concept and the effectiveness of the design method. To sum up, the proposed perturbation approach by using the additional metalized via-holes in dual-band or wide-band even tunable band SIW filters offers a number of advantages including: 1) high selectivity of dual band or wide band, 2) low-cost fabrication using the printed circuit board technology, 3) easy integration with the single-layer circuits and systems, 4) convenient implementation of the tunable or reconfigurable band, and 5) enhanced power capability and decreased radiation loss due to complete shielding with standard metalized via-holes.In order to construct a new single-layer dual-band or wide-band dual-mode SIW filter, a novel perturbation approach, which uses additional metalized via-holes, is proposed in this paper. Unlike the previous methods in the papers678fm0n is the resonant frequency of the TEm0n mode, c0 is the light velocity in a vacuum, εr is the relative dielectric constant of the chosen substrate, Lc is the length (width) of the single square cavity, dw is the diameter of wall via-holes, sw is the diameter of wall via-holes, and m and n are the indices of the modes. In addition, to avoid dispersion loss of typical SIW structures, the parameters of the wall via-holes are required to meet the following condition in practical applicationsfm0n of the resonant modes TEm0n and the values of necessary parameters including dw, sw, m and n are known, the initial sizes of the square cavity can be readily determined using 101 and TE102 modes, respectively. The three-dimensional (3D) and planar two-mode electric-field vector distribution in a single cavity are illustrated in z-axis, and parallel to the x-axis) cannot affect the electric-field vector distributions of the TE102 mode, because the center of the cavity is equivalent to a short-circuited point for the TE102 mode. In contrast, the electric-field vector distribution of the TE101 mode is influenced obviously by these additional via-holes. The significance of the flat distribution of the TE101 mode with perturbation via-holes shown in In this proposed dual-band or wide-band dual-mode SIW filter, the first and the second passbands are generated by the TEf1 and f2 are the first and second cavity resonant frequencies, respectively. The results depicted in 102 mode, but directly changes the field distribution and the corresponding scattering-parameter performance of the TE101 mode. Therefore, the first band can be controlled and tuned by applying different numbers of perturbation via-holes without affecting the second band. Note that when the number of via-holes increases to twelve, two different bands generated from both TE101 and TE102 modes are combined into a single wide band from 6.06 GHz to 6.40 GHz , as depicted in slotL) and width (slotw) are presented in slotL and slotw as shown in 101 and TE102 modes have shifted marginally by less than 1.5%, which can be neglected or easily corrected in practical circuits and systems.In order to fabricate the reconfigurable dual-band dual-mode SIW filters, adding the required slot around the perturbation via-holes is a popular option from practical implementations point of view. 2. As can be seen, there is a good agreement between the simulated and measured results. Considerable deviations are observed in the measured results compared with the simulated ones, which can be attributed to finite conductivity of the metal/via, the fabrication error, the degradation caused by the SMA(SubMiniature version A) connectors. As the number of perturbation via-holes increases from 1 to 8, the measured lower frequency f1 shifts from 4.71 GHz to 5.63 GHz, thus indicating wide-range frequency tunability for f1. We have presented the realization of a new wide-band dual-mode SIW filter with 12 perturbation via-holes, as shown in Subfigures of etc. Due to the reconfigurable feature of 4 perturbation via-holes with different states, the fabricated SIW filter has 16 states to control the final dual-band performance. State 0000, State 1000 and State 1111. As shown in 101 mode, while the disconnected ones do not affect the electromagnetic distribution of this mode. 102 mode. 101 mode) is tuned from 4.61 GHz to 5.24 GHz, whereas the measured higher frequency (the resonant frequency of the TE102 mode) is unchanged at about 6.18 GHz. The simulated and measured curves are consistent with the explanation made in 2. These results confirm and verify the operation of a novel dual-band dual-mode SIW filter with the independently reconfigurable TE101 resonant mode.101, but the second resonant mode TE102 is not affected. Based on the analysis of the operating principle and sensitivity studies of parameters, four two-cavity dual-band SIW filters and a reconfigurable two-cavity dual-band SIW filter are designed, simulated, fabricated and theoretically and experimentally verified. The measured results indicate that the available measured frequency-ratio ranging from 1 (wide-band case) to 1.3  can be realized in four two-cavity dual-band SIW filters, and the center frequency of the first band can be tuned from 4.61 GHz to 5.24 GHz but the center frequency of the second one will be unaltered and fixed at near 6.18 GHz in the two-cavity dual-band SIW filter with four reconfigurable via-holes. In summary, the proposed perturbation approach by using the additional metalized via-holes in dual-band or wide-band even tunable band SIW filters offers several advantages including 1) high selectivity of dual band or wide band, 2) low-cost fabrication using printed circuit board technology, 3) easy integration with single-layer circuits and systems, 4) convenient implementation of tunable or reconfigurable band, and 5) enhanced power capability and decreased radiation loss due to complete shielding with standard metalized via-holes. The fixed or tunable dual-band filters can be used to construct a multi-mode and multi-band filter array. In addition, it can be used in a Cognitive Radio System, an FM Communication System and a Radar RF System. This paper provides a method for designing a dual-band SIW filter with a tunable lower passband and a fixed upper passband, and the frequency ratio of dual-band can be adjusted regularly. It can be applied in the particular dual-band transceiver system that the higher passband needs to be fixed while the lower passband can be switched flexibly. Nevertheless, for a system requiring arbitrary dual-band frequencies, the fixed band can be obtained by altering the overall dimensions of this SIW filter, while the other band can be obtained by utilizing the method proposed in this paper. Hence, the proposed method is universal for certain system frequencies. In conclusion, this work provides not only a solution for designing a dual-band SIW filter, but also universal strategies and thoughts for realizing reconfigurable filters.This paper presents a novel realization of dual-band or wide-band dual-mode substrate integrated waveguide (SIW) filters based on additional metalized via-holes. The main feature of the proposed approach is that the independent perturbation can be introduced to the first resonant mode TEHow to cite this article: Wu, Y. et al. Dual-Band Dual-Mode Substrate Integrated Waveguide Filters with Independently Reconfigurable TE101 Resonant Mode. Sci. Rep.6, 31922; doi: 10.1038/srep31922 (2016).