JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.Metatarsals are frequent sites of stress and fragility fractures in younger athletic populations and aging older adults. Metatarsal fractures are particularly common in Charcot neuroarthropathy (CN), a complication of diabetes mellitus (DM) and peripheral neuropathy (PN). Neuropathic metatarsal fractures may be caused by an accelerated cortical bone osteolysis and may be reflected as geometric‐derived strength estimates from standard foot radiographs. The purpose of this cross‐sectional study was to determine geometry and strength‐derived estimates of the metatarsals in individuals with DM, PN, and CN compared with younger and older adult controls who were nondiabetic and nonneuropathic. We studied 62 participants: 20 young adult controls (YACs), 22 older adult controls (OACs), and 20 diagnosed with DMPN&CN. From weight‐bearing radiographs, we measured the outer diaphysis diameter and inner marrow diameter at the distal, middle, and proximal diaphysis sites of the second and fifth metatarsal. From these diameters, we derived strength estimates of combined cortical width (CCt.Wi), percent cortical area (%Ct.rA), buckling ratio (BR), moment of inertia (MOI), and section modulus (SM) at each site in both metatarsals. DMPN&CN participants had an accelerated cortical thinning, decreased %Ct.Ar, increased BR, and lower MOI and SM compared with OACs and YACs. The OACs showed age‐related decreases in CCt.Wi and % Ct.Ar, and increased BR. The BR demonstrated significant group × bone × site interaction with the distal fifth metatarsal in the DMPN&CN group having the lowest bone strength. The BR in the distal fifth metatarsal of DMPN&CN participants was 36% and 49% greater than in the OAC and YAC groups, respectively. DMPN&CN participants have lower metatarsal bone strength estimates compared with younger and older adult controls. Standard foot radiographs demonstrate an accelerated cortical osteolysis in DMPN&CN individuals, particularly in the distal fifth metatarsal diaphysis. © 2019 The Authors.  Human pedal bones, particularly the metatarsals, are common sites of fracture because of the high loading, bending, and twisting forces that occur during weight‐bearing while walking, running, and jumping, as well as the rapid changes in direction required in activities of daily living and recreation.By contrast, human metatarsals are comprised largely of cortical bone throughout the entire length of the diaphysis; therefore, they may be a more appropriate choice to determine cortical osteolysis that accompanies bone pathologies and their associated risk for metatarsal fracture. Although standard foot radiographs often can reveal diffuse patterns of low bone mass (osteopenia) or osteoporosis in select pedal bones, radiographs are generally not recommended to quantify the magnitude of bone loss in either the cancellous or cortical compartments because of their lack of precision.Despite several studies reporting a rapid pedal osteolysis over short periods resulting in an elevated risk for new or recurrent fracture in Charcot neuroarthropathy (CN),Our underlying premise was that cortical osteolysis would manifest as a deterioration in metatarsal diaphysis geometry and strength‐derived indices, which is accelerated by DMPN&CN. We selected the second metatarsal because it represents the longitudinal midline axis of the foot; is commonly the longest, most‐dense metatarsal in the human foot; and is often a site for stress‐related fracture.The study was conducted at Washington University School of Medicine  and was approved by its Human Research Protection Office's Institutional Review Board. Participants were recruited through local advertisements; written informed consent was obtained from each participant. Participants were remunerated for their participation. Briefly, eligibility criteria included: (1) for young adult control (YAC) participants, an age <45 years and without a current or recent 1‐year history of foot or ankle impairments or deformities; (2) for older adult control (OAC) participants, an age ≥55 years without DM or PN or major midfoot and hindfoot deformities; and (3) as the primary index group, participants with an acute or subacute stage of CN, with a diagnosis of DM (type 1 or 2) with Semmes‐Weinstein monofilament‐confirmed evidence of PN.Three groups of participants were studied in a cross‐sectional comparison of radiogrammetric‐derived geometric and strength parameters of the second and fifth metatarsal bones at each of three diaphysis sites . Standard weight‐bearing dorsoplantar AP radiographs of at least one foot were assessed by raters who were blinded to one another. Radiographs of only the affected foot of the DMPN&CN group were used and values were averaged from radiographs of both feet (when available) in the YAC and OAC groups. All radiographs were uploaded and imported into ISite PACS  software program for measurement. The ISite PACS software measures angles to the nearest 1‐degree increment and distance measures to the nearest 0.1‐mm increment. A calibration ruler was included in each weight‐bearing radiograph to correct for any magnification error.Two novice raters performed all the radiogrammetric measurements of metatarsal geometry. Each novice rater underwent instruction, training, and practice independently of one another. Raters made measures independent of one another and were instructed not to discuss the results with each another.To assess intra‐ and interrater reliability, each rater measured the same 30 radiographs on two separate occasions with a 3‐ to 14‐day interval between repeated measures. Radiographs for intra‐ and interrater reliability were randomly selected from all (right and left feet) available radiographs by one of the authors (KLB) from participant numbers assigned from all three groups of participants included in the study. Both raters were blinded to group allocation of radiographs and blinded to each other's measures throughout the study. Each rater assessed and recorded the length of each metatarsal as the distance between lines drawn tangent through the extreme points of each metatarsal. Each rater determined the proximal (approximately 65% to 75%), middle (50%), and distal (25%) distances from the distal (head) region to make measurements of cortical geometry ; (4) site buckling ratio , and (5) the section modulus . At the distal, middle, and proximal diaphysis of both metatarsals, we modeled each metatarsal as a circular annulus with 100% of the cortical bone area at a constant radius from the center of the circle to the outer cortical  edge. The CCt.Wi was calculated by subtracting the Ma.Dm from the B.Dm at each of the three sites along the diaphysis.B.Dm/2)2– (π · (Ma.Dm/2)2)/(π · (B.Dm/2)2] × 100.ro4 – ri4], where ro = outer bone radius and ri = inner marrow radius. The cross‐section MOI was used to calculate the section modulus (mm2), calculated as MOI divided by the outer bone radius (ro). The BR for each of the three diaphysis sites was computed as the ratio of the outer bone radius (ro) divided by the average single cortical wall width (½ CCt.Wi).From the geometry measurements, we calculated cortical geometry and bone strength parameters.Participant characteristics were compared using one‐way ANOVAs or Fisher exact tests. We determined the intra‐ and interrater reliability from estimates of intraclass correlation coefficients (ICC) using fixed effects model for each of the two novice raters. The primary dependent variables included in the intra‐ and interrater reliability analysis included the measured variables‐ metatarsal length, B.Dm, and Ma.Dm at each of the three sites on each metatarsal as parameters of metatarsal geometry. Because all bone strength parameters were calculated from the geometric variables, no reliability estimates were required.p value for an effect was significant, follow‐up comparisons were conducted to isolate the nature of the effect. For main effects, all pairwise comparisons were examined. For interactions, we took a simple main effects approach. Pairs of means for one of the variables involved in the interaction were compared within each level of the other variable involved in the interaction (or each combination of levels if the interaction is three‐way). Follow‐up comparisons were adjusted using the Holm procedure to control the type I error rate at .05. Dependent variables tested included B.Dm, Ma.Dm, CCt.Wi, %Ct.Ar, MOI, BR, and SM at the proximal, middle, and distal diaphysis sites of the second and fifth metatarsal bones. When assumption tests occasionally indicated violations of either normality, homogeneity of variance–covariance matrices or sphericity, we conducted randomization tests to verify all inferences. Data are presented in the tables as adjusted mean ± adjusted SD. An alpha level ≤0.05 was significant. All statistical analyses were performed in R version 3.5.1We used a 3 × 2 × 3 (group × bone × site) fixed effect analysis of covariance (ANCOVA): with group treated as the between‐subject factor, bone and site treated as the within‐subject factors, and body weight included as a covariate. Group differences were not adjusted for age because age accounted for group variation by design. The ANCOVA group main effect tested our primary null hypothesis of no group differences in metatarsal geometry or derived‐strength estimates. Within the framework of the ANOVA when the Participant characteristics are shown in Table All groups differed significantly in geometric and strength‐derived estimates Tables  and 4. PThere were differences in geometric and strength‐derived estimates between the metatarsals with the fifth metatarsal having lower CCt.Wi and %Ct.Ar and higher Ma.Dm and BR compared with the second metatarsal. MOI and SM were not different between the second or fifth metatarsals.Geometric and strength‐derived estimates were different at all diaphysis sites. For MOI, B.Dm, and SM, the distal site was lower compared with the middle site, which in turn was different from the proximal sites. For CCt.Wi, the middle and proximal sites were lower than the distal site, but not different from each other. For %Ct.Ar, the distal site was lower than the middle site, which was lower than the proximal site. For BR, the middle site was lower than the proximal site, which was lower than the distal site. For Ma.Dm, the middle site was lower than the distal site, which was lower than the proximal site.There was a group × bone interaction for CCt.Wi, %Ct.Ar, and BR. Interactions were evaluated in two ways: First, bone differences were tested within each group, and then group differences were tested for each bone. For CCt.Wi, there were differences between the second and fifth metatarsals in the OAC and DMPN&CN groups, but not the YAC group. The second approach indicated group differences for the second metatarsal; the DMPN&CN group was different from the YAC and OAC groups, which were not different from each other. For the fifth metatarsal, all groups differed from one another. For %Ct.Ar, there were significant bone differences for the DMPN&CN and OAC groups, but not the YAC group. Also, for the second metatarsal, the DMPN&CN group was different from the YAC, but not the OAC group. The control groups were not different from one another. For the fifth metatarsal, all groups were different from one another. For BR, there were bone differences for the DMPN&CN group, but not for either control group. For group differences tested for each bone, the second metatarsal in the DMPN&CN group was different from the YAC group, but not different from the OAC group. The control groups were not different from one another. In the fifth metatarsal, all groups were different from one another. There were no group × bone interactions for B.Dm, Ma.Dm, %Ct.Ar, MOI, or SM.There were group × site interactions for Ma.Dm and BR. These interactions were expressed in two ways: Sites were tested within each group, and then group differences were tested for each site. The first approach for Ma.Dm showed that all diaphysis sites were different from each other in the YAC and OAC groups. In the DMPN&CN group, the Ma.Dm at the proximal site was different from the middle and distal sites, which were not different from each other. The second approach showed that for the Ma.Dm distal site, none of the groups was different, but the middle site in the YAC group was different from the OAC and DMPN&CN groups, which were not different from each other. For Ma.Dm at the proximal site, the OAC group was different from the YAC group, but not from the DMPN&CN group. The DMPN&CN group was not different from the YAC group. Using the first approach of testing site differences within each group, we found that for the BR all sites were different from all other sites for both control groups. For the DMPN&CN group, the distal site was different from the middle and proximal sites, which were not different from each other. Using the second approach of testing for group differences at each site, the BRs in all groups were different from each other for the distal site. For the middle site, the DMPN&CN group was different from the YAC and OAC groups, which were not different from each other. For the proximal site, DMPN&CN group was different from the YAC group, but not different from the OAC group. There were no differences between the control groups. There were no group x site interactions for B.Dm, CCt.Wi, %Ct.Ar, MOI, or SM.There were bone × site interactions for B.Dm, Ma.Dm, CCT.WI, %Ct.Ar, and BR, but not for MOI or SM. These interactions were expressed in two ways: first, testing for site differences within each bone, and then testing for bone differences at each site. For B.Dm, we found significant bone differences at the distal site, and for both the second and fifth metatarsals, each site was different from one another. For Ma.Dm, there were significant bone differences for the middle and proximal sites, but not for the distal site; for the second metatarsal, each site was different from each other. For the fifth metatarsal, middle and distal sites were different from the proximal site, but not different from each other. For CCt.Wi, the first approach found there were bone differences for the middle and proximal sites, but not for the distal site. The second approach found that for the second metatarsal, the distal site was different from the middle and proximal sites, which were not different from each other, whereas for the fifth metatarsal, all sites were different from each other. For %Ct.Ar and BR, the first approach showed there were bone differences for the middle and proximal sites, but not for the distal site. For the second and fifth metatarsal, each site was different from each of the other sites.p = .06) group × bone × site interaction, where the DMPN&CN's distal site BR on the fifth metatarsal averaged 4.5 (highest), resulting in the lowest resistance to fracture  in the outer  and inner (marrow) diaphysis diameters in both the second and fifth metatarsals. The periosteal expansion in B.Dm represents a well‐known homeostatic compensatory adaptation in the aging of long bones that compensates for the age‐related osteolysis at the cortical envelope, but maintains the areal cross‐sectional MOI and SM and attempts to preserve the resistance to fracture.Additionally, this is the first study to report the intra‐ and interrater reliability estimates of radiographic‐derived indices of metatarsal geometry by novice raters. The intra‐ and interrater ICC reliability estimates were high for all sites along the second metatarsal and distal and middle diaphysis sites in the fifth metatarsal. The exception to high reliability estimates was the proximal end (base) in the fifth metatarsal, where interrater ICC estimates were poor . The likely explanation for the poor interrater ICC estimates at the proximal base of the fifth metatarsal is that there was much overlap with the fourth metatarsal base overlapping with the fifth metatarsal base on the AP radiograph, therefore obscuring the outer and inner bone diameters on the radiographs. Each rater clearly measured their same proximal region of the fifth metatarsal at time 1 and again at time 2 as demonstrated by high intrarater ICC values; however, the proximal site may have differed markedly in location between each rater accounting for poor interrater agreement at the proximal site.There have been previous studies of human metacarpalsThe utility of assessing geometric strength indices of the metatarsals, particularly the BR and SM, has been previously verified. Gutekunst and colleagues demonstrated that BR and SM at the middle site of the second and third metatarsal calculated from volumetric quantitative CT scans accounted for 89% of the variance in ex vivo failure loads using three‐point mechanical bending. BR and SM as geometric strength indices, in combination with density‐weighted BMD, were highly predictive of ex vivo metatarsal failure loads.The results from our radiogrammetric study of human metatarsal cortical bone loss are consistent in magnitude with what we have observed using quantitative ultrasonometry in the calcaneus in subjects with acute CN.There are limitations to our study. Foremost, the design of our current study did not allow us to discern the distinct contributions of disease (diabetes), neuropathy, or acute CN on metatarsal osteolysis. To directly address the distinct contributions of each, additional control groups including non‐Charcot neuropathic and nonneuropathic participants will need to be included. Our previous work suggests that an acute or prolonged inflammation associated with CN is largely the incipient contributor of the 5% to 6% cortical osteolysis in the metatarsalsAnother limitation is when estimating the metatarsal strength indices, we assumed the metatarsals to be uniformly circular with a constant diameter (and radius). Similarly, we assumed the cortical width (and area) around the marrow cavity was uniform and therefore can be represented by a single average cortical width. Although these assumptions allowed us to quickly derive and estimate the structural indices as described by Beck and colleagues for increasing fragility of the hip,increases in diaphysis diameters with decreases in cortical geometry , resulting in significant increases in BR at the distal, middle, and proximal diaphysis of the second and fifth metatarsals in participants with DMPN&CN. In comparison, smaller adaptations in cross‐sectional MOI and SM at the metatarsal shafts were observed in the DMPN&CN and OAC groups to maintain resistance to bending, despite structural adaptations that accompanies aging. Cortical osteolysis and an increasing BR are easily identified and assessed using standard digital foot X‐ray radiogrammetry. An increasing BR may signal an increasing risk for stress‐related and fragility‐induced metatarsal fractures.In summary, our results show an accelerated cortical osteolysis in feet of individuals with DM, PN, and CN compared with older adults. These changes arise from All authors state that they have no conflicts of interest.


Aspergillus parasiticus and Aspergillus niger was investigated. AhCPI showed a concentration-dependent antifungal activity against both fungi. Differential effects were observed when fungi were treated with cystatin in two developmental stages. When AhCPI was added to young mycelium cultures of A. niger, it had a dramatic effect on mycelial growth compared with old mycelium cultures. On the contrary, there was no differential effect of AhCPI addition to either old or young mycelium of A. parasiticus. Furthermore, electron microscopic observations showed that cystatin caused important effects at the level of cell morphology and organelle integrity of both fungi. Additionally, A. parasiticus spores treated with AhCPI presented sensitivity to oxidative, osmotic and ionic stresses; in opposition, under same conditions, A. niger did not show sensitivity to any stressful agent. These results suggest that AhCPI antifungal activity might be related with damage to cell integrity, affecting the survival of the fungi. In addition, our evidences showed that fungal species respond dissimilarly to cystatin; however, such disparities can be used to the control of unwanted fungi.The inhibitory effect of recombinant amaranth cystatin (AhCPI) on the spore germination and growth of the mycotoxigenic fungus  The antifungal activity against these fungi has not been analysed, even though the mycotoxigenic fungi A. flavus and A. parasiticus represent a hazard to agricultural production, human health and animal production . AhCPI was expressed in ion Zain . Whereas2.2.1.E. coli cells as well as its purification by affinity chromatography was done according to the protocol reported by Valdés-Rodríguez et al. (Escherichiacoli M15 (pREP 4) cells containing the amaranth cystatin coding sequence were grown at 37°C until they reached an OD600 of 0.5. The expression of cystatin was induced at 37°C by the addition of 0.1 mM Isopropyl beta-D-1-thiogalactopyranoside, and the cells were harvested by centrifugation at 17,300 × g for 10 min after 5 h of incubation. From cell lysates, AhCPI was purified using an affinity nickel resin column, previously equilibrated with phosphate buffer. The cystatin was eluted by 250 mM imidazol dissolved in the same buffer. The purified cystatin was exhaustively dialysed against water in a microdialysis system.The isolation of recombinant AhCPI from transformed z et al. . Escheri2.2.Aspergillus parasiticus, American Type Culture Collection (ATCC) 16,992 (aflatoxin producer) and Aspergillus niger ATCC 1015 (acid organic producer – GRAS) . Fungal strains were grown in potato dextrose agar  for 6 and 4 days, respectively. Conidia were harvested by washing the mycelium colony with 10 mL of water plus Triton X-100 solution (0.01%), and counted with a haemocytometer to adjust to the desired inoculum concentration with sterile distilled water. Before inoculation, conidia suspensions were kept overnight at 4°C to ensure optimal germination. In order to analyse the effect of AhCPI on mycelial growth, spore suspensions (≈1000 viable spores) of A. niger and A. parasiticus were cultivated on potato dextrose broth (PDB) media at 28°C with addition of different concentrations of AhCPI  in 250 μL as total volume in a microtiter plate. After different periods of incubation, fungal growth was measured by absorbance at 492 nm in a microplate spectrometer . The assays were performed with 12 biological replicates.The antimicrobial toxicity of AhCPI was assayed with two different types of fungi: 2.3.3 spores mL−1) were grown in microtiter plates in absence of AhCPI under the same conditions described earlier. Spores were allowed to grow for 28 and 43 h before different concentrations (20 µM in the case of A. niger and 30 µM in the case of A. parasiticus) of the AhCPI were added to the cultures. Microtiter plates were incubated for additional 44 and 29 h, respectively, to reach 72 h in all treatments. Samples from the different conditions were taken for microscopic observations.Spore suspensions of each fungus (1 × 102.4.9 spores in 60 µl of water plus 0.01% triton) was inoculated separately in 4 ml of PDB and incubated at 28°C. Then AhCPI at final concentration of 10 µM and 20 µM was added to the A. niger and A parasiticus spore suspensions. No AhCPI was added to the control cultures of both fungi. Flasks containing the treatments were incubated for 65 h at 28°C, and samples were taken for microscopic observations.Fungal spore suspensions were incubated at 4°C overnight. Afterwards, a spore suspension of each fungus  of A. niger and A. parasiticus were incubated for 96 h at 28°C in PDB with 15 and 20 μM of AhCPI, respectively, in microtiter plates. These concentrations of AhCPI were selected because fungal growth was affected at the same level (5 spores mL−1 (counted with a Neubauer chamber), 10-fold serial dilutions were prepared, and 10 μL of each fungus was spotted on PDA plates with addition of different compounds: 1% H2O2; 0.05 mM Rose bengal; 30 mM LiCl; 2 M Sorbitol. Fresh spores incubated previously at 4°C overnight and not treated with AhCPI were used as control. Plates were incubated at 28°C for 72 h and photographed with a Cannon camera model Eorebeld.Spores , and increase in absorbance was indicative of spore germination and mycelial growth. At higher concentrations of cystatin, there was inhibition of spore germination, since absorbance measurements were lesser . Regarding A. parasiticus, the fungus showed higher tolerance to AhCPI treatment than A. niger, and higher concentrations of AhCPI were tested. Concentrations of 20 and 25 μM of AhCPI inhibited 23.2% and 61.2% of fungal growth, respectively, while 30 μM inhibited 82.79 % (data not shown). The effective concentration for 50% growth inhibition (EC50) was about 12.75 µM for A niger and 21 µM for A. parasiticus. It is interesting to observe the wide range of concentrations in which AhCPI affects different fungal species. The reported EC50 in Fusarium oxysporum was 13 μM; in Slerotium cepivorum, it was 0.38 μM, and in Rhyzoctonia solani, it was 0.16 μM  inhibited the mycelial growth of Claviceps paspali, Claviceps purpurea, Curvularia fallax, Curvularia cymbopogonis, Curvularia lunata, as well as Alternaria solani and Fusarium oxysporum  was able to inhibit mycelial growth and cause the lysis of sclerotia in Sclerotium rofsii (Yang & Yeh 2005). The recombinant barley and chestnut cystatin inhibited the growth of Botrytis cinerea, Colletotrichum graminicola, Septoria nodorum and Plectosphaerella cucumerina; interestingly, it did not affect Trichoderma viride . At this point, it is important to mention that the filamentous fungus A. niger is highly resistant to osmotic and ionic stresses (Wucherpfennig et al. The electron microscopic evidence presented in this study strongly suggests that AhCPI antifungal activity over c stress . Aspergith AhCPI  and B. O4.A. parasiticus, and the GRAS fungus A. niger by inhibiting spore germination and mycelial growth. Additionally, this study showed some evidence that AhCPI provokes impaired fungal cell integrity, and in A. parasiticus, sensitivity to stressful conditions.These results confirmed the antifungal activity of AhCPI against the mycotoxigenic fungus No potential conflict of interest was reported by the authors.


Ns) metric indicate that loess particles at saturation with respect to liquid water conditions are marginally more efficient than the mineral and soil dust values reported in the literature, but they have lower efficiencies than the predicted Ns efficiency of K-feldspar particles at supercooled temperatures greater than -38 °C.The heterogeneous nucleation of ice processes involving loess particles that influences the formation of mixed-phase clouds are poorly understood. Here, the ice nucleating ability of wind-blown dust or loess accumulated from the past glaciated area was investigated at three temperatures: -26, -30, and -34 °C and at below and above saturation with respect to liquid water conditions. Total six loess samples from different regions across Columbia Basin province, WA, USA were collected, dry dispersed, size-selected at mobility diameter 200 nm, and investigated for their ice nucleation efficiency. To understand the effect of atmospheric processing during long-range transport on their ice nucleating ability, similar experiments were also performed on acid-treated loess samples. Additionally, the ice nucleating properties of Arizona Test Dust (ATD) were investigated as a surrogate for natural mineral dust particles to test the experimental approach. Results show that treated particles have lower ice nucleation efficiency compared to untreated particles at all temperature and saturation with respect to liquid water conditions. Comparison based on ice-active site density ( Silt-sized terrestrial sediment or loess formed by the accumulation of wind-blown dust covers approximately 10% of the world’s land surface . Loess dPrevious studies concluded that individual, as well as a complex mixture of different, minerals are efficient ice nucleating particles (INPs) in different modes of ice nucleation , 11. HetIn this paper, we report the first results from an experiment designed to investigate the ice nucleation efficiency of bare and acid coated loess from Columbia Plateau province of the northwestern United States. To understand the implications of this loess variability towards ice nucleation, bulk surface samples were collected from six sites distributed across the plateau. These samples were size-selected based on their mobility diameter, and investigated for their ice nucleation efficiency at various temperatures and humidity conditions. To understand the effect of atmospheric aging, particularly chemical aging where condensation of acids may occur while particles are transported in the atmosphere, the samples were treated with acids, and the ice nucleation efficiency of these acid treated particles was investigated.Six undisturbed surface samples of loess from Columbia Plateau province were collected. No specific permissions were required for these locations because they are public. This field study also did not involve endangered or protected species. The location of the sampling site was based on the map that shows loess distribution across the province , 16. SeeLoess samples were sieved (Newark Superla) to eliminate any vegetation debris and particles larger than ~50 μm. ice) and relative humidity with respect to water (RHw) using Murphy and Koop (2005) . Retrieved July 3, 2019 from (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Ice melt contributes 39%  of the total streamflow increase. However, streamflow from the western slopes is projected to decrease by 5% , due to the smaller contribution of ice melt, less precipitation and higher evapotranspiration. Increasing water supply from the eastern slopes suggests more water availability for currently degraded downstream ecosystems in the Xinjiang province of China, while the likely decreasing streamflow in Central Asian rivers on the western slopes indicates new regulations will be needed.The glacierized Tien Shan – Pamir – Karakoram mountain complex supplies water to about 42 million people. Yet, the knowledge about future glacial runoff in response to future climate is limited. Here, we address this issue using a hydrological model, that includes the three components of glacial runoff: ice melt, snowmelt and the runoff of rainfall over ice. The model is forced by climate projections of the CMIP5 models. We find that the three components exhibit different long-term trajectories, sometimes opposite in sign to the long-term trend in glacier impacts. For the eastern slope basins, streamflow is projected to increase by 28% ) by the late 21 Along the Western slope of the divide, the major rivers Amu Darya (WAM), Syr Darya (WSY), and Chu River (WCH) flow to Central Asian countries, Tajikistan, Kyrgyzstan, Uzbekistan and Turkmenistan, supplying over 90% of the water resources for 22 million people in Central Asia2. The Ili River (WIL) originates in Xinjiang China and flows to the lake Balkhash in Kazakhstan. Along the Eastern slope of the divide, the Junagr river system (EJG) includes rivers originating from northern side of the eastern Tian Shan and flowing into the Jungar Basin; the Kaidu River (EKD) flows into the Boston Lake, and the Weigan River (EWG), Tailan River (ETL), Tarim River (ETR) and Kashgar River (EKG) flow into the Tarim Basin. The ETR consists of the Aksu River originating from the middle Tien Shan, the Yarkant River from Karakoram, and the Hotan River from the western Kunlun mountain. These rivers from the eastern TPK contributes to the water supply for 20 million people in the Xinjiang province, and sustains downstream ecosystems, which are currently degraded3. Changes in the magnitude or seasonality of runoff may thus have important consequences downstream to both human society and ecosystems. From inventories (see Supplementary Information), the study area is covered 27,101 glaciers with a total area of 36,539 km2 (see Table The Tien Shan-Pamir-Karakoram (TPK) mountain complex is the “water tower” of the Central Asian countries east of the Aral Sea , and the Xinjiang province of western China see Fig.  that was6. Glacier retreat in response to this warming has been observed in the Tien Shan8 and Western and Central Pamir10, affecting water availability17. In contrast, some regions of the Karakoram, Eastern Pamir and Western Kunlun show stable glaciers or even mass gain23. This local response has been attributed to various causes such as the reduction of mean summer temperatures24 and the low sensitivity of snowfall to warming in winter25.Evidence for recent climate change, given by local meteorological records over the past five decades, shows a warming rate in a range of 0.1–0.42 °C per decadeet al.4 and Unger-Shayesteh et al.5, there is a lack of integrated assessment of the impacts of climate change on the future evolution of the TPK glaciers and their consequences for river discharge. One difficulty in assessing river discharge in this region is the unknown future behavior of glacial runoff, which is considered in this study as the sum of ice melt, snowmelt and the runoff of rainfall over ice26. These three processes have distinctly different responses to climate change. Ice melt has a long-term response to climate change, while the supraglacial snowmelt and rainfall runoff over ice have much faster changes than it. Because ice melt is difficult to measure directly for a larger area26, we used an glacier - enhanced SWAT (Soil Water and Assessment Tool) model27 to simulate it. This model (see Supplementary Information) describes glaciers as hydrological response units (GHRUs) and calculates ice melt based on glacier mass balance and an area-volume scaling relationship29. A GHRU is divided into elevation bands of equal intervals, and the mass balance of a glacier is the sum of the balance of each band. It also calculates the supraglacial snowmelt and the runoff of rainfall over ice components of glacial runoff. The GHRU approach has been incorporated into the distributed Soil Water Assessment Tool (SWAT)31 to simulate catchment hydrological changes. We calibrated and validated the combined glacier-hydrology model using daily (in Xinjiang basins) and monthly  streamflow records from 55 gauging stations  and temperature data from PGMFD . For the simulated and observed monthly streamflow of those ten rivers, the Nash-Sutcliffe efficiency criterion34 varies between 0.33 and 0.94 with a mean of 0.79 and standard deviation (SD) of 0.11; the relative bias ranges between −30 and 23% with a mean of 2% and SD of 9%; the coefficient of determination of a linear regression between observed and modelled discharge over the gauged period 1961–2007 ranges from 0.77–0.94, with a mean of 0.87 and SD of 0.06 in the ten studied rivers. Linear regression between the simulated and observed glacier area change has a correlation coefficient of 0.97 with a coefficient of determination of 0.91 and zero intercept for the ten basins considered  were derived for each RCP at the 10th and 90th percentiles of the projected temperature and precipitation changes, and the model runs closest to the percentiles were eventually selected36 . On the eastern slope EWG, ETL, EKG, and ETR river basins, which are heavily glacierized, the glacial-runoff contribution to annual streamflow ranges between 40 and 61%, while the ice melt contributes 19 to 22%. Glaciers in this region are usually located in high elevation headwater sub-basins so that the glacial-runoff and ice-melt contribution ratios decrease as the river progresses downstream14. For example, in the WAM on the western slope, the glaciers are concentrated in the Pyanj and Vakhsh tributary sub-basins, where the glacier-runoff contribution to the annual streamflow of the whole basin represents less than 13%, and the ice-melt contribution 7%. The fractions of meltwater runoff and rainfall-runoff in streamflow for the ten rivers in Fig. The retrospective analysis of the period 1966–1995 indicates a recent mean glacial-runoff 35. However by 2095 under RCP8.5 coupled with continued global increases in greenhouse gases and temperatures, the mean glacier-area loss will become larger than in RCP2.6, with a 70 to 81% (mean 79%) loss on the western slope, and 52 to 93% (mean 59%) on the eastern slope. Meanwhile, in the RCP8.5 climate scenario, by 2095, relative to the 1960s, the loss of ice-water storage ranges between 46 and 94% (mean 54%) on the eastern slope and 64 to 68% (mean 65%) on the western slope; in two river basins, EKD in the eastern slope and WCH in the western slope, most of the glaciers will have disappeared. Glaciers decrease less on the eastern slope because they experience a larger increase in precipitation, and because their average size is greater, they are less vulnerable to warming. Details of projected glacier changes are presented in Fig. The model estimated that in the future both glacier area and ice-water storage will decline significantly, yet with regional differences. Relative to the 1960s under the low-warming scenario RCP2.6, between 36 and 55% (mean 52%) of the glacier area is projected to disappear by 2045 in the western slope basins, compared to 19 to 59% (mean 26%) loss for the eastern slope. Among all the scenarios, RCP2.6 projects the smallest increase in temperature; however, the TPK glacier area will continue to decrease in the twenty-first century, especially in its second half, indicating that glacier retreat will be a general trend under atmospheric warming in the future. Under the high and continuous warming scenario of RCP8.5, the glacier-area loss is similar to RCP2.6 by 2045, given the small differences in temperature and precipitation at that time between both scenarios17. They indicated that in the glacier fed Aral Sea (Amu and Syr) and Tarim rivers, annual glacier runoff is projected to rise until roughly the middle of the century, followed by steadily declining glacier runoff thereafter under RCP 4.5. For the Tarim River, the projected long-term patterns are compatible. However, for the WAM and WSY, we projected that the turning points have already passed  were compared see Figs  and S3. see Fig. . The difFuture changes in streamflow were presented in Fig. very likely40 (94% probability) to increase in 2066–2095  than increase, when all the uncertainties of climate models and scenario differences are considered.As a result of changes in glacial runoff and changes in precipitation in the non-glacierized parts of each catchment, streamflow is projected to increase in eastern slope rivers (and in WIL), but to decrease in western slope rivers between 1966–1995 and 2066–2095. For RCP2.6, streamflow increases in eastern slope catchments by 8%  between 1966–1995 and 2066–2095; for RCP8.5, streamflow also increases by 28% , with ice melt contributing 39%  of this signal. Taking into account the range of future uncertainties resulting from climate models and scenario differences, the streamflow in the Xinjiang rivers is see Figs . For theUnder RCPs 2.6, 4.5, 8.6 and relative to 1965–1995, the streamflow is expected to change by 6.5–8.3 billion cubic meters (bcm) for Xinjiang rivers and (−)0.6–1.2 bcm for Central Asian rivers in 2045–2066, respectively. In 2066–2095, the change is expected to be 3.1–11.0 bcm for Xinjiang rivers and (−)5.3 – (−)0.8 bcm for Central Asian rivers, respectively see Fig. .By comparing long-term changes in the water balance components, i.e., precipitation, change in ice storage, and evapotranspiration (see Supplementary Information), we found that increasing ice melt and precipitation are the dominant reasons for streamflow increase in rivers on the eastern slope. In the heavily glacierized basins of the EWG, ETL, and ETR, where today’s glacier coverage is above 5% and ice melt makes up over 13% of the streamflow, the future increase in streamflow is dominated by loss of ice-water storage see Figs . In the Figure Changes in hydrographs due to snowmelt, ice melt, and streamflow are more apparent for rivers on western slopes than on eastern ones see Fig. . The sno36. In some of the TPK tributary catchments, the streamflow is projected to increase until the 2020 s but then begins to decline until the end of the twenty-first century15. It is worth noting that this study projects similar long-term trend patterns of melt water and streamflow in those tributary catchments, but with later turning points and slower decline in glacier area, melt, and streamflow up to the end of the twenty-first century. This projected behavior is possibly due to uncertainties caused by the glacial melt modelling approach and the GCM scenarios.In the contiguous region of the TPK, ranging from the south slope of Karakoram to Himalaya, runoff in the twenty-first century is expected to increase consistently due to increasing glacier melt and precipitation until the 2050set al. (2011) evaluated quite a few data products in the Hymalays waersheds and indicated that APHRODITE data, a product processed from gauge stations, which gives the best precipitation estimates when compared to independent ground observations41. They also indicated that the lack of stations at high elevation limits the accuracy of this data set; more ground-based observations could allow for better conclusions drawn from any atmospheric-related studies in the Hymalas region; this might be also correct for the Tien Shan – Pamir – Karakoram Range.As most of the similar studies in the high mountain regions, scarcity of the ground-based climatic data is always an important issue to deal with. Different proxy data base may be used to drive the simulation. Uncertainty caused by the precipitation data remains an unaddressed issue. Andermann Evapotranspiration is another important water balance component which might be a source of uncertainties. It depends upon inputs of the historical and future climate, and the theoretical approaches (see Supplementary Information). However, evapotranspiration is perhaps a grayer zone than other water balance items in the alpine hydrology due to data scarcity in data for input and for output evaluation of the theoretical approaches.Populus Euphratica forests growing along the lower reach of the Tarim River3. However, the fact that streamflow will not increase, or will more likely decrease, in the WCH, WSY and WAM rivers indicates that human use of water should not exceed current levels if we are to avoid risks of trans-boundary water disputes between Central Asian countries. Earlier shifts in the streamflow hydrographs and reduction of the high-flow volume in the WSY and WAM rivers also implies that water regulations should be improved in Central Asian countries, to allow hydropower generation in upper-reach countries (Kyrgyzstan and Tajikistan) and summer irrigation in the lower-reach countries 2.In summary, glacier mass loss in response to climate change will lead to contrasting water supply situations for the downstream users of TPK water resources. The increase in water supply on the eastern slope will favour water users downstream in Xinjiang during the next decades, albeit with a significant loss of ice. This extra water may provide an opportunity to restore the degraded riparian forest ecosystems, e.g., the 27 to simulate glacier processes and dynamics. The model is suitable for the analysis of large river basins. The GHRU approach explicitly simulates glacier mass balance and transient area–volume dynamics interactively on a time-step of one day. Each GHRU is divided into 100 m elevation bands, to account for spatial variation in precipitation and temperature over the altitudinal range. Glacier mass balance is simulated for each elevation band. The mass-balance components included are: the accumulation of snow, transformation of snow to ice, sublimation/evaporation, melting and refreezing. In the model, supraglacial snowmelt is calculated first, and then ice melt if the snow disappears leaving ice exposed to air temperature. The model neglects the effects of debris-cover found on some glaciers. Both snowmelt and ice melt were calculated using degree-day factors. Rain falling on to ice is assumed to run off directly as “runoff of rainfall over ice”. A volume-area scaling relationship was used to simulate the transient change of glacier area and volume, updated by ice mass changes. In contrast to other glacio-hydrological models that lump glaciers and glacier patches in an individual grid square into a single virtual glacier43, the GHRU approach simulates individual glaciers and therefore can capture the different transient climate-change responses of glaciers of different sizes. Glacial runoff was separated into ice melt, supraglacial snowmelt, and runoff of rainfall over ice.We used a fully distributed glacio-hydrological model, applying a GHRU approach31 hydrological model to simulate basin-wide glacio-hydrological processes. The glacier-enhanced SWAT model simulates glacial and hydrological processes and routes water through the channel systems to the outlet.The GHRU approach was incorporated into the SWAT44, and the first (1960s) and second (2010s) Chinese glacier inventories8. A total of 27,101 glaciers were modelled, for details of each basin refer to Table Glacier-area data were compiled from the World Glacier Inventory (WGI) data set released by the National Snow and Ice Data Center (NSIDC), the Tien Shan and Pamir glacier inventories of Aizen32, and daily maximum and minimum temperatures for 1948–2008  from PGMFD33. Post-processing of these data sets is described in the Supplementary Information.The forcing meteorological data came from meteorological stations, daily precipitation data for 1951‒2007  from APHRODITEThe glacier-enhanced SWAT model was calibrated and validated using daily or monthly streamflow records from 55 gauging stations and the basin-wide glacier-area change in the Manas, Kaidu, Weigan, Tailan, Kashgar and Tarim basins on the eastern slope, and the Ili, Chu, Syr and Amu basins on the western slope of the TPK mountain complex see Fig. , which s35 2.6, 4.5 and 8.5. Within the CMIP5 model space, we constructed four composite contrasting climate change trajectories for each of the five sub-regions of the TPK Annual catchment water balances included the input precipitation, the outputs of evapotranspiration and streamflow at the outlet, and the storage changes in ice, soil moisture, and groundwater. The simulation results indicated that the annual storage changes of soil moisture and groundwater over the long term are negligible. Thus the storage-change terms reduce to ice change only.ETa is derived from the potential evapotranspiration (PET) for all land use and land cover types in this study. The SWAT model provides several approaches for estimating PET, and we used the Hargreaves (H-G) method31. The H-G method is temperature based, i.e., it needs only the daily maximum, minimum, and mean temperatures as input. Meanwhile, it takes into account of effect of solar radiation on PET by inclusion of the extraterrestrial radiation. The extraterrestrial radiation depends only upon solar declination, geographic latitude, and the angular velocity of earth’s rotation. There is not any parameter in the H-G method that needs calibration and validation based on historical data series, while the historically based parameter might not be suitable for future climate as discussed in literatures47. Application of the H-G method is also found in similar studies in Karakoram – Himalayan range watersheds49.The Comparing changes of the water balance components averaged over 2016–2045 and 2066–2095 to that over 1966–1995, we identified the patterns of hydrological variables and the reasons for streamflow changes over the long term. Further details of the methods used are given in the Supplementary Information. Supplementary Information


The purpose of this study was to present a surgical technique for taking out universal screw and nail caps which were difficult to removed. We used a variety of industrial hex wrenches, dental drills, and other equipment to take out internal hex nuts with different specifications (32 pieces) and universal screws (15 pieces) in 28 patients. A total of 32 nuts were taken out, 3 of which were polished by the industrial drill. A total of 17 were spun by hand, 2 were spun by locking pliers, 10 were turned by “I” type screwdriver, and 3 were turned by bone blade. A total of 15 screws were taken out, 9 of which were removed with a wrench and the other 6 by means of locking pliers after re‐fixing with a truncated titanium rod. The novel technique is simple and provides a solution following failure of a supporting device. The pedicle screw internal fixation system is widely used for the treatment of spinal fractures and degenerative instability10–T12 in 3 cases, T11–L1 in 4 cases, T12–L2 in 8 cases, L3–L5 in 5 cases, and L4–L5 in 8 cases. Fractures had healed and bone graft fusions were intact in all patients.The 28 patients in this study were aged between 33 and 62 years, with an average age of 43 years. Among them, there were 22 men and 6 women, 16 with spinal fractures and 12 with degenerative diseases of the spine. The operation time ranged from 10 to 83 months after the last operation. Internal fixation sites: TAll patients were operated on in prone position under general anesthesia, and internal fixation screws were exposed through conventional posterior midline incision. This study focused only on the screw removal procedure.A wide variety of industrial‐sized hex wrenches Fig.  were steA wrench was selected that best matched the hexagon in the groove. It was placed in the groove at the end of the screw to loosen the screw. If the hexagonal groove in the tail of the screw had slid or was covered by bone tissue, we cut the titanium rod which had been removed earlier to approximately 1 cm with a rod breaker device, which is a commonly used instrument for spinal internal fixation surgery, and selected a nut to fix the 1‐cm titanium rod firmly in the groove of the end of the screw Fig. , consoliAll screws were completely removed. All patients in this study were followed up, with a follow‐up time between 11 and 24 months, and an average follow‐up time of 13 months. All the postoperative incisions healed well. All patients were followed up with X‐ray and CT examination after surgery, indicating good bone healing at the nail tract, no foreign body residue, no pain in the spine with good activity, and no complications of infection and nerve injury.The method is easy to implement and is a novel technique that has not been described previously in the published literature. It provides a solution following failure of a supporting device. However, grinding the nail will prolong the operation time. If a new type rod of breaker instrument that is similar to titanium mesh scissors can be designed, the titanium rod could be cut off directly by the tail of the screw without increasing soft tissue damage.Attention should be paid to the application process: (i) the inner hexagon wrench should be placed at the lowest position of the nut groove and it is best matched with the inner hexagon, and rotating the wrench should not be done with force to avoid causing the screw groove to slip; (ii) when grinding the nut with the dental drill from the inside out, use its side to grind the groove wall of the nut or tilt the drill from the top down, and choose between the two arms of the nail tail; (iii) the industrial hexagon wrench and dental drill should be updated timely to avoid screwing pedical screw nut slippery and breaking dental drill when grinding; and (iv) when grinding the two sides of the nut, the upper and lower openings of each side should be as wide as possible, and the screwdriver or bone blade should be changed if necessary during the grinding process to avoid openings that are too wide on both sides of the nut.


Uncertainty in the RADARSAT-2 approach may be linked to unexplored questions about scattering mechanisms and the interaction of radar signal with mid-latitude lake ice. The application of optimized temperature models to a long-term temperature record revealed a thinning of ice cover by 0.81 cm per decade.Lakes are a key geographical feature in Canada and have an impact on the regional climate. In the winter, they are important for recreational activities such as snowmobiling and ice fishing and act as part of an important supply route for northern communities. The ability to accurately report lake ice characteristics such as thickness is vital, however, it is underreported in Canada and there is a lack of lake ice thickness records for temperate latitude areas such as Central Ontario. Here, we evaluate the application of previously developed temperature models and RADARSAT-2 for estimating lake ice thickness in Central Ontario and provide insight into the regions long term ice thickness variability. The ALS Environmental Science Shallow Water Ice Profiler (SWIP) was used for validation of both temperature and radar-based models. Results indicate that the traditional approach that uses temperatures to predict ice thickness during ice growth has low RMSE values of 2.3 cm and correlations of greater than 0.9. For ice decay, similar low RMSE values of 2.1 cm and high correlations of 0.97 were found. Using RADARSAT-2 to estimate ice thickness results in R Lakes are an important feature of the Canadian landscape and hold 7% of the world’s freshwater resources . The preLake ice thickness has historically been estimated using a combination of non-linear and linear temperature models e.g. –10). Ste. Ste10])In order to estimate ice thickness using the temperature-based model, climate data is needed from local weather stations, unfortunately, the distribution of temperature stations is sparse throughout regions of Canada and is not representative for estimating ice thickness across large areas. Satellite remote sensing provides an alternative to these temperature-based methods and can cover large spatial areas, allowing for ice thickness measurements at the regional scale. Passive microwave satellite observations have been used to estimate ice thickness e.g. ,17), how, how17])This study addresses the paucity of in situ lake ice thickness records in the mid-latitudes through testing the applicability of both temperature based and remote sensing based ice thickness models. Specifically, the objectives of this study were to; i) estimate lake ice thickness in Central Ontario using established temperature models, ii) determine the applicability of using SAR from RADARSAT-2 for estimating ice thickness in Central Ontario, and iii) investigate long-term trends in ice thickness for Central Ontario.2 with a maximum depth of 39.6 m )Ice thickness estimates for the 2016–2017 ice season from both the temperature and backscatter models are a good representation of the values that were obtained from the SWIP , Fig 8. th and February 3rd). However, the HH model is more affected by surface conditions than the temperature model causing over and under estimates. On February 19th, the HH model underestimated the ice thickness by 13.26 cm. This was due to a period of warmer temperatures that induced melt and increased water content in the snow; the increased water content would have absorbed the incoming beam from the RADARSAT-2 satellite resulting in the lower backscatter observed for the day, -16.8 dB. This can also be seen in th and March 2nd (denoted by triangles) are underestimated compared to SWIP data and the estimated temperature-based ice thickness. The under estimation during this time is due to the change in surface conditions , which was captured by shoreline observations. Additionally, on February 12th, the HH model overpredicts ice thickness (43.58 cm) compared to the closest field measurements for that day (33.05 cm) as a result of erroneously high backscatter (-12.84 dB) from the previously mentioned mix of ascending and descending orbits from the radar images.Comparisons between these estimated data, SWIP measurements, and field observations  show thaUsing the daily homogenized Canadian surface air temperature data (1888–2017), we developed a long-term estimate of maximum ice thickness for MacDonald Lake using the combined optimized AFDD model and ATDD model. There is a general trend toward lake ice thinning in Central Ontario at a rate of 0.81 cm per decade (p < 0.01) , howeverIce thickness trends in the Arctic  are more extreme with decreases in ice thickness modelled between 21 and 38 cm 1950–2011) and 18–22 cm (1991–2011) depending on snow cover  and 18–2,56. LongβF in the first model, when a γ of 0.5 is used, was found to be 1.73. This value is within the range, 1.7–2.4, that has been noted in other literature for a lake with average snow cover [The original Stefan’s model and the optimized AFDD model were found to accurately estimate ice thickness when compared to the SWIP and manual measurements. The optimized value for ow cover ,15.βF value dependent on surface conditions [βF and γ values as done in this study. Lebedev [βF value of 1.33 and a γ value of 0.58 for sea ice in the Arctic near Russia, while an equation developed for Button Bay near Churchill, Manitoba used a βF value of 1.53 and a γ of 0.59 [βF values for these studies are higher than what we found in Central Ontario for MacDonald Lake, the γ values are very similar (0.60), showing that the optimized AFDD model proposed for Central Ontario fits within the accepted literature. The use of this optimized model improved results compared to the modified Stefan’s Eq (γ of 0.5 is used), resulting in lower RMSE when applied to the validation data, confirming that this method is appropriate for estimating ice thickness.Although the modified Stefan’s Eq  typicallnditions ,15, the  Lebedev , used a  of 0.59 . AlthougIce thickness estimates were also accurately estimated during melt season when using the maximum ice thickness determined by the optimized AFDD model and an empirical cut-off of 90 ATDD to set the end of the ice season. The resulting estimates of ice thickness were strongly correlated with SWIP measurements with a Kendall Tau-b of 0.97 and a RMSE of 2.1 cm. The optimized AFDD model and the ATDD model can be combined in order to provide a complete record of thickness throughout the ice season, and the full season record was shown to have a high index of agreement (0.93) when compared with SWIP measurements. The combination of these models is the best choice when temperature data are available and phenology dates can be determined from satellite or other observation data. When no phenology data are available and only temperature data exists, the modified Stefan’s model (1) can still be used, with the caveat that the freeze timing based on AFDD alone may be too early in temperate latitudes. Future work could investigate the possibility of using a delay based on temperature during the ice-free season to more accurately represent the start of freeze in these latitudes. For larger regions where in situ temperature data are too sparse, the use of finer resolution gridded temperature data (e.g. ERA5 from ECMWF), or satellite-based temperature data (MODIS-LST), may be a viable option for driving the degree day models. The suggestion of using satellite-based temperature data supports the applicability of remote sensing to cover large spatial areas  and prov2 = 0.6, RMSE 11.7 cm), it does provide an alternative method to the degree-day models for isolated lakes where accurate temperature data may be unavailable, particularly for the initial growth period. On the other hand, the proposed model lacks some of the predictive strength observed in previous literature, e.g. R2 = 0.99 using C-band FMCW system in laboratory conditions [2) compared to MacDonald Lake (~1.6 km2) [nd, 2016 to February 13th, 2017), found a significant relationship with a high R2 of 0.66 and a lower RMSE compared to the model developed for the full ice season when all data points are used (6.8 cm vs. 11.1 cm). This shows that HH backscatter may have the most applicability during the early stages of ice growth.Although there is a higher amount of error for the model based on HH backscatter (Rnditions . The dif1.6 km2) ,58. ReseIn order to improve the results of the HH model, further investigation into how temperate latitude lake ice affects HH backscatter is needed. Additionally, there is a need for a better understanding on how the initial formation of snow ice impacts backscatter. Snow ice has also been previously reported as being a large contributor to scattering in temperate latitude lake ice due to the large number of bubbles found in this layer ,59. SnowOverall, lake ice thickness is more accurately estimated in Central Ontario using temperature models compared to models based on HH backscatter. Both the modified Stefan’s equation and optimized AFDD model were able to accurately predict ice thickness based on measured SWIP data. However, the optimized AFDD model does require in situ or satellite observations to capture the start of freeze onset to increase accuracy. The equations are presented as a simple method that can be used to estimate ice thickness for areas where nearby temperature data are available. This could be quite beneficial for communities that rely on ice thickness for tourism and recreation to supplement their manual measurements, or who may be interested historical thickness trends for their area (with the caveat that thickness maybe overestimated without including the freeze-up timing). Furthermore, the identified cut-off of 90 ATDD for water clear of ice could be used to improve automated methods of identifying ice phenology from remote sensing images by providing an estimate for the end of the ice season. RMSE values from the optimized AFDD model and ATDD model were 2.3 cm during ice growth and 2.1 cm during ice decay with correlation statistics greater than 0.9 for both ice growth and decay. The combined AFDD and ATDD model was also used to show that ice thickness for MacDonald Lake, and likely all small/medium sized lakes in Central Ontario, has been thinning since temperature records began there in 1888.This work also raises the question of whether the observed HH backscatter increase is a result of single-bounce scattering from the roughening of the ice-water interface over time, as determined for northern lakes , rather S1 FileThe supplementary data file includes metadata information about the location of the weather station at MacDonald Lake, full temperature data from the weather station for 2015/2016 and 2016/2017 ice seasons, and SWIP recorded ice thickness data for 2016/2017.(XLSX)Click here for additional data file.


A-receptor resulting in sedation, anxiolytic or anti-convulsive effects. Flavonoids of several classes are inhibitors of monoamine oxidase A or B, thereby working as anti-depressants or to improve the conditions of Parkinson’s patients. Flavanols, flavanones and anthocyanidins have protective effects preventing inflammatory processes leading to nerve injury. Flavonoids seem capable of influencing health and mood.Flavonoids are present in almost all terrestrial plants, where they provide UV-protection and colour. Flavonoids have a fused ring system consisting of an aromatic ring and a benzopyran ring with a phenyl substituent. The flavonoids can be divided into several classes depending on their structure. Flavonoids are present in food and medicinal plants and are thus consumed by humans. They are found in plants as glycosides. Before oral absorption, flavonoids undergo deglycosylation either by lactase phloridzin hydrolase or cytosolic β-glucocidase. The absorbed aglycone is then conjugated by methylation, sulphatation or glucuronidation. Both the aglycones and the conjugates can pass the blood-brain barrier. In the CNS several flavones bind to the benzodiazepine site on the GABA Throughout time, many plants have been used for the treatment of mental problems. A good number of those are alkaloid-containing plants, as alkaloids are known to interact strongly with receptors in the central nervous system, but in recent years it has become clear that flavonoids may also play a role in enzyme- and receptor systems of the brain, exerting various effects on the central nervous system, including prevention of the neurodegeneration associated with Alzheimer’s and Parkinson’s diseases. Matricaria recutica L. — Asteraceae) have for centuries been used for their calming effect, which is due to apigenin , after which plasma and urine was collected over a period of 24 h [O-glucuronidated, methylated and sulphate conjugates of epicatechins and epigallocatechins and the unmetabolized flavan-3-ols (-)-epigallocatechin-3-O-gallate and (-)-epicatechin-O-gallate. Detectable quantities of the flavan-3-ols were present in the plasma 30 min after consumption of the green tea. The predominant metabolite detected in plasma was epigallocatechin-3-O-glucoronide, reaching a Cmax of 126 nmol/L and a Tmax of 2.2, while the lowest amount detected was of (-)-epicatechin-3-O-gallate with an Cmax of 25 nmol/L and a Tmax of 1.6 h. In another study human subjects were given a single dose of 20 mg/mL green tea solids dissolved in warm water [O-gallate, (-)-epigallocatechin and (-)-epicatechin revealed Cmax values of 78 ng/mL, 223 and 124 ng/mL , respectively. The time to reach Cmax values was in the range of 1.3-1.6 h. Corresponding AUC values were 508.2, 945.4 and 529.5 ng h mL-1 respectively.In a human study, ten healthy subjects were asked to consume 500 mL of green tea which contained 648 µmol of flavan-3-ols [mainly, (-)-epigallocatechin-3- of 24 h . Analysirm water . SubsequO-glucoside and the 3,4’-O-glucoside [O-sulfate, quercetin-3-O-glucuronide, isorhamnetin-3-O-glucuronide, quercetin-O-glucuronide-O-sulfate and quercetin-O-diglucuronide. The principal metabolites were quercetin-3’-O-sulfate  and quercetin-3-O-glucuronide . The lowest Cmax (62 nmol/L) was detected for quercetin-O-diglucuronide, with a Tmax of 0.8 h. The bioavailability of quercetin-3-O-rutinoside was studied in human subjects given a single dose of 176 µmol of the quercetin rhamnose-glucose disaccharide in the form of 250 mL tomato juice [O-glucuronide and isorhamnetin-3-O-glucuronide with Cmax values of 12 nmol/L and 4.3 nmol/L, respectively. For both metabolites a Tmax value of around 5 h was observed. In a human feeding study, subjects were given an acute dose of 275 µmol flavonol glucosides in the form of fried onions with the main constituents being the 4’-lucoside . Analysito juice . In thisO-rutinoside and naringenin-7-O-rutinoside was investigated in human volunteers given 250 mL orange juice which corresponded to a dose of 168 µmol and 12 µmol of the two flavanones, respectively [O-glucuronide and a hesperetin-O-glucuronide, which was not further assigned. The total Cmax value of the two metabolites was 922 nmol/L with a Tmax ca. 4 h. In a similar study where the bioavailability of hesperetin and naringenin was investigated in human subjects given a dose of 444 mg hesperetin and 96 mg narirutin in 1 L orange juice [The bioavailability of the flavanones hesperetin-7-ectively . Two metge juice , peak plIn a human study investigating the bioavailability of the pure isoflavones genistein and daidzein, volunteers was given a dose of 0.8 mg/kg administered in gelatin capsules . SubsequO-glucoside and small amounts of pelargonidin-3-O-rutinoside (13 µmol) and cyanidin-3-O-glucoside (6 µmol) [O-glucoronide, and three other pelargorinidin-glucoronides and a pelargorinidin-rutinoside in amounts that was not quantifiable. The peak plasma concentration of pelargorinidin-O-glucoronide was 274 µmol/L at a Tmax of about 1 h. In a human study, subjects was given 200 g of strawberries containing 222 µmol pelargonidin-3-(6 µmol) . Plasma The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells, however other cell types such as pericytes, astrocytes and neuronal cells also play an important role . Brain cin vitro study two aspects of the interaction between flavonoid molecules with the BBB was examined: the uptake of selected flavonoids, and their glucuronides into two brain endothelial cell lines (b.END5 and RBE4) and their permeability across an in vitro model of the BBB (ECV304 cells co-cultured with C6 glioma cells) [In an a cells) . The twoin vivo study rats were given a standardized Ginkgo biloba extract (EGb761®) containing 22-27% flavonoid glycosides, mainly glycosides of quercetin, kaempferol and isorhamnetin [In an in vitro studies.The content of flavoniods in brain tissue is given per mass of total protein analyzed . This value cannot easily be used for estimates of intracellular molar concentrations of flavonoids, making it difficult to compare values with results obtained from in vivo models, where effects on the CNS have been demonstrated. The flavonoid glycosides linarin, 2S-hesperidin, 2S-neohesperidin, 2S-naringenin, diosmin, gossipyn and rutin were found to exert a depressant action on the CNS of mice following i.p. injection, measured in the hole board, thiopental induced sleeping time and locomotor activity tests [in vivo [Flavonoids have been investigated in ty tests . The agl[in vivo ,29. On t[in vivo . Passiflora coerulea L.(Passifloraceae) increased the number of entries into and time mice spent in the open arms in the elevated plus-maze test of anxiety, consistent with an anxiolytic action. Chrysin also increased the time spent on head-dipping. In contrast, chrysin had no myorelaxant action, indicating that chrysin possesses anxiolytic [Scutellaria baicalensis Georgi (Lamiaceae), which is used for sedation, showed selective sedative and anticonvulsant activity in vivo, which support a subtype specific activity on the GABAA-receptor of the flavone [A-receptor [Chrysin, a flavone from xiolytic . The fla flavone . Epigallreceptor ,34.in vivo studies show that flavonoids are able to be absorbed after oral administration, pass the blood-brain barrier and do have various effects on the CNS.The A receptors. GABAA receptors are heteromeric GABA-gated chloride channels. The transmembrane ion channel is opened by a stimulus generated by GABA, which allows an influx of chloride ions. This results in a decrease of the depolarizing effects of an excitatory input, thereby depressing excitability [A receptor consists of five subunits, made up of two α, two β and one γ or δ subunit. Several isoforms exists , potentially giving a vast number of combinatorial mixes. However, only ten subunit combinations make up the physiologically relevant GABAA receptors in the brain [GABA is the most important inhibitory neurotransmitter in the human central nervous system. GABA is involved in epilepsy, sedation and anxiolysis, and works via binding to GABAtability . As a rehe brain ,37.Besides the binding site for the neurotransmitter itself, there are modulatory binding sites on the receptor. Benzodiazepines bind to the so-called benzodiazepine site, where they modulate the receptor to be more sensitive to GABA, and thereby yielding an anticonvulsant, sedative or anxiolytic effect. A-benzodiazepine site have been isolated from plants. Apigenin was isolated due to the compound’s affinity to the benzodiazepine site from Matricaria retutica (Asteraceae) [Tanacetum parthenium (Asteraceae) [Sersia dentata Thunb. (Anacardiaceae) [i-values have been reported: 3 μM [Valeriana wallichii D.C. , and had a Ki-value 495 nM [Artemisia herba-alba Asso (Asteraceae), and IC50-values of 1.3 µM, 23 µM, 104 µM and 8 µM were reported, respectively [Passiflora coerulea (Passifloraceae) with a Ki-value of 3 µM [A number of monoflavonoids with affinity to the GABAeraceae) , Tanaceteraceae)  and Sersdiaceae) . A numbeed: 3 μM ,40, 4 μMed: 3 μM , 9 μM [2ed: 3 μM . 6-methye 495 nM . Dinatinectively ,43. Chry of 3 µM .S)-naringenin, isolated in a bioassay-guided isolation from Menthaaquatica L.(Lamiaceae), had a high IC50-value of 2.6 mM [The active monoflavononoids isolated from plants are all flavones. The flavanone ,Hypericum perforatum L. (Clusiaceae) and Searsia pyroides Burch. (Anacardiaceae). It has been shown to exhibit high affinity to brain benzodiazepine receptors in vitro [i values of 6 nM [Sersia pyroides inhibited 3H-flumazenil binding with a Ki of 82 nM [Searsia pyroides extract, as well as apigenin, amentoflavone and agathisflavone, showed inhibitory effects on spontaneous epileptiform discharges in mouse cortical slices of the plant extract, but no effects of the isolated flavonoids, indicating that the anticonvulsant effect is due to involvement of a different neurotransmitter system [The biflavonoid amentoflavone is found in a number of plants with medicinal properties, including in vitro , with Ki of 6 nM , 7 nM [3 of 6 nM ,40 and 5 of 6 nM . Studies of 6 nM . The bifof 82 nM . Furtherr system . A receptor where flavonoids bind. Electrophysiological studies have indicated that there might be a high-affinity flumazenil-sensitive site, and another low-affinity, flumazenil-insensitive site. Further, it has been shown that apigenin, hesperidin and (-)-epigallocatechin gallate enhance the activity of the clinically used benzodiazepine diazepam [Interestingly, there might be two binding sites on the GABAdiazepam ,51, by aIn vivo, 6-hydroxyflavone exhibited anxiolytic effects, without sedation, cognition impairment, motor incoordination or convulsions [Some flavonoids have been shown to have subtype specific activity. It is reported that 6-hydroxyflavone acted as a subtype selective partial positive allosteric modulator at the flumazenil sensitive benzodiazepine site. 6-hydroxyflavone had stronger affinity to receptors containing α2 and α3-subunits, than to receptors with α1 and α5-subunit. vulsions . FurtherMonoamine oxidase (MAO) is a flavoenzyme found in the outer membrane of mitochondria. MAO catalyzes the oxidative deamination of primary, secondary and some tertiary amines . Two isoGingko biloba extract by means of HPLC [50 values of: kaempferol (0.7 µM), apigenin (1 µM), chrysin (2µM) and quercetin (5 µM). Phenelzine, a non-selective and irreversible inhibitor of MAO was used as a reference compound . Quercetin was isolated from heather  Hull – Ericaceae) and identified as a MAO-A inhibitor with an IC50 value of 18 µM. In the same assay clorgylin, a selective MAO-A inhibitor, had an IC50 value of 0.2 µM [50 value of 0.01 µM for MAO-A and 20 µM for MAO-B [Melastoma candidum D. Don (Melastomataceae) were shown to inhibit MAO-B with IC50 values of 19, 12, 4, 11 µM, respectively, in an assay where deprenyl (a selective MAO-B inhibitor) had an IC50 value of 19 µM [Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) and found to inhibit MAO-B with IC50 values of 89 and 59 µM, respectively, while deprenyl had an IC50 value of 0.3 µM [Sophora flavescens Ait. (Fabaceae) exhibited monoamine oxidase inhibitory activity: formononetin an isoflavone with IC50 values of 21 µM (MAO-A) and 11 µM for MAO-B and the flavanone kushenol F with IC50 values of 104 µM (MAO-A) and 63 µM for MAO-B [Mentha aquatica L. (Lamiaceae) in a bioassay-guided fractionation process [50 value for MAO-A inhibition was 955 µM and 288 µM for MAO-B in an assay where the IC50 value of clorgylin was 0.0003 µM and 0.1 µM for deprenyl. In a recent study the inhibitory effects of pure anthocyanidins on MAO-A and MAO-B activity was investsigated [50 values were obtained for MAO-A and MAO-B inhibitory activity, respectively: malvidin (22 µM and 19 µM), pelargonidin (27 µM and 43 µM), cyanidin (30 µM and 32 µM), peonidin (31 µM and 22 µM), petunidin (32 µM and 43 µM), delphinidin (35 µM and 31 µM). In the same study different glycosides and diglycosides of the above mentioned anthocyanidins were also studied with IC50 values in the range of 29-117 µM for MAO-A inhibition and 31-242 µM for MAO-B inhibition. Several flavonoids have been identified as inhibitors of MAO-A and MAO-B. The flavonols kaempferol and quercetin and the flavones apigenin and chrysin were isolated from a standardized  of HPLC . All fouor MAO-B . Quercetof 19 µM . The flaf 0.3 µM . Two flaor MAO-B . Naringe process . The IC5All of the active flavonoids identified in the above possess inhibitory activity on MAO-A, MAO-B or both. Furthermore, this inhibitory activity is not confined to a single flavonoid class as all the classes presented in figure 1 are represented. Evidence is mounting that flavonoids have effect on memory, cognition and neurodegeneration. Although many of the studies in this field have been performed with flavonoid-rich fruits, both these studies and studies with isolated flavonoids indicate that flavonoids have potential to protect neurons against injury induced by neurotoxins and neuroinflammation, a potential to activate synaptic signalling and an ability to improve cerebrovascular blood flow, as has recently been reviewed .O-methyl-epicatechin and hesperetin protected neurons against oxidative neuronal damage [Studies with fruit supplements indicate that flavanols, flavanones and anthocyanidins have the capacity to improve memory. (-)-Epicatechin has been shown to improve rat spatial memory in the water maze test . It is tl damage ,68. The l damage .The flavl damage ,71. A-receptor, producing sedation, anxiolytic or anticonvulsive effects. Flavonoids of several classes are inhibitors of MAO-A and -B, thus mediating an anti-depressant or anti-Parkinson’s activity. Dietary flavonoids are absorbed as aglycones from the intestinal lumen and subsequently conjugated in the enterocytes to glucuronides, sulphates and methylates of the parent aglycone. Thus, flavonoids are present in the plasma almost exclusively in a conjugated form. Flavonoids, both as aglycones and in the conjugated form pass the blood-brain barrier. During passage of the blood-brain barrier conjugates may be metabolized back to the parent aglycone, which then enters the central nervous system. In most studies the compounds isolated from the plant e.g., aglycones and glycosides, were tested, where as the effect of conjugation are rarely considered. In the brain flavanoids act on different systems. Flavanols, flavanones and anthocyanins may act in protective ways, increasing the cerebral blood flow and protecting the neurons against inflammatory processes leading to cell injury. Flavones may interact with the GABAWith this background it seems plausible that the diet may influence a person’s health state and even mood. Plants that are rich in flavonoids of the flavanol, flavanone and anthocyanin-type are berries, citrus fruits, apple, pear, tea, cocoa and wine, which are common food products. It should be noted that the plants rich in GABA-active flavones are not common food plants, but fall within the category of medicinal plants. It thus seems that over time humanity have found plant products suitable for consumption as functional foods, and others that are left for medicinal purposes.


The Antarctic ice sheet is one of the largest potential contributors to future sea level rise. Predicting its future behaviour using physically-based ice sheet models has been a bottleneck for the past decades, but major advances are ongoing. Unlike atmospheric models, ice sheet models emerged very recently. The first numerical Antarctic continental-scale ice sheet models, for instance, saw the light at the beginning of the 1990s. Initially, such models were employed at rather coarse resolution (∼50 km) to investigate ice sheet changes during glacial-interglacial cycles. At that time, ice sheets were believed to be a slow component of the climate system with a highly diffusive response to palaeo-climatic changes. This diffusive nature stems from the fact that ice sheet models were based on the so-called 'Shallow-Ice Approximation (SIA)', which is the dominant type of ice deformation for a large ice sheet resting on a near-frozen bed and based on the premise that ice deformation is due to shearing close to the bed. This may be valid for the bulk of ice sheet flow, but breaks down near its edges. It results in a gradual, slow response to the imposed climatology with a time lag that increases with the size of the ice sheet due to its internal thermomechanics (cold ice deforms slower than temperate ice).1, hypothesizing a possible collapse of the West-Antarctic ice sheet (WAIS) as a consequence of anthropogenic global warming2. The WAIS is a marine ice sheet for which the bed lies well below sea level. As the ice thins towards the edge of the ice sheet, ice thickness becomes equal to the buoyant thickness of ice and starts to float, forming ice shelves. The contact where ice starts afloat is called the grounding line. The bed is also depressed deeper in the centre of the ice sheet, making the bed slope inland from the grounding line to the centre, hence creating a reverse or retrograde bed slope. The proposed instability, known as Marine Ice Sheet Instability  of the WAIS4.MISI theory was challenged by most ice sheet models, as they considered that ice shelves were too weak  to affect the force balance of the grounded ice sheet. This led to the hypothesis of neutral equilibria that were neither stable nor unstable5, dispelling the previous idea that ice sheets and ice shelves are mechanically uncoupled. This had a profound impact on ice sheet model development, pushing models to conform to known  solutions, which led to international model intercomparisons, such as the Marine Ice Sheet Model Intercomparison Project (MISMIP)6. MISMIP allowed for a collective improvement of marine ice sheet models by adapting both their physical basis as well as their numerical approaches. Besides being the limit of flotation, a grounding line is also the change from a shear-dominated ice flow to an ice flow dominated by longitudinal pushes and pulls (due to membrane stresses). The transition is never sharp, but gradual, and largely depends on the processes that govern basal motion under ice sheets within fast-flowing ice streams . These membrane stresses need to be resolved across the grounding line with a sufficiently high spatial resolution, which pushed the development of spatial grid refinement in numerical models through sub-grid interpolation schemes, the use of unconstructed grids or adaptive mesh approaches7. It also requires the use of approximations other than the SIA to the flow of ice, ranging from the Shallow-Shelf Approximation to full-Stokes models, which are harder to solve.Mathematical proof of the MISI theory put forward by Weertman was finally given three decades later5 remains only valid for unconfined ice shelves, i.e. ice shelves that do not exert a force on the inland ice sheet other than the (ocean) water pressure. In reality, however, ice shelves are bounded within embayments, thereby exerting a back force to the grounding line, hence limiting the flow speed of ice streams and their discharge of ice through the ice shelf into the ocean. This is commonly known as the buttressing effect of ice shelves that act as a cork on a bottle, preventing the ice to flow out too fast. Loss of ice-shelf buttressing has effectively been witnessed in Antarctica at the beginning of the 21st century, where ice shelf collapse in the Antarctic Peninsula led to increased glacier discharge8.Despite these theoretical and numerical advances, the developed MISI theory9. This eventually leads to ice shelf collapse if sufficient melt water and cracks are available. Hydro-fracturing is also considered a precursor for a new emerging mechanism in ice sheet modelling, the concept of Marine Ice Cliff Instability  and calving front/cliff stability still need to be further explored. While such mechanisms aid at explaining past changes in the Antarctic ice sheet, they do show a higher sensitivity to forcing, and hence lead to a significant larger mass loss10.Ice shelf weakening happens through both atmospheric and ocean processes, such as hydro-fracturing and sub-shelf melting. Hydro-fracturing is a process that increases the water pressure from surface melt on ice shelves in surface crevasses, thereby widening them so that they become more vulnerable to calving11. As with calving due to hydro-fracturing, sub-shelf melting decreases the buttressing capacity of ice shelves via loss of pinning points and weakening of the ice shelf through thinning. This has presumably been the trigger of the observed acceleration of large Antarctic outlet glaciers in the ASE during the last decade. However, sub-shelf melt is particularly determined by ocean circulation within the ice shelf cavity, which, in turn, requires high-resolution ocean circulation models to link large-scale ocean circulation to sub-shelf melt. While such ongoing and future developments require increased computer power, intermediate solutions can be obtained through physically based parametrisations of sub-shelf melt based on sub-shelf ocean circulation12.Sub-shelf melting is responsible for more than half of the ice mass loss at the margins of the Antarctic ice sheet13. Moreover, using a steady-state for initialising the ice sheet prevents models from properly accounting for the dynamical mass losses observed over the last decade, as the present-day ice sheet is not in steady state. Motivated by the increasing ice sheet imbalance of the ASE glaciers over the last 20 years, and supported by the recent boom in satellite data availability, data-assimilation methods are progressively used to evaluate unknown fields using time-evolving states accounting for the transient nature of observations and the model dynamics14.A key aspect of projecting future Antarctic mass loss with dynamical ice sheet models is related to the initial state of the model. Since ice sheet models were initially applied for palaeo-climatic studies on long time scales, initialisation was generally obtained from a long spin-up time leading to a steady-state ice sheet (both in terms of geometry and thermodynamics). However, for predictions on shorter time scales (decades to centuries), a stable spin-up generally leads to an ice sheet geometry far different from the one currently observed, which is one of the reasons why such ice sheet models may respond differently than observations suggest15. The increase in computational efficiency enabling high spatial resolution modelling, high-resolution datasets of bedrock topography and surface velocity, longer time series on ice sheet changes, and the improved initialisation of ice sheet models are now allowing ice sheet modelling to move away from the slow-diffusive response over millennium time scales towards robust predictions on decadal time scales, hindcasting and potentially reanalysis.Antarctic ice sheet modelling has taken a big step forward based on an improved understanding of key processes and the development of assimilation methods leading to the ability to reproduce observed ice sheet changes and by making refined future projections. From a theoretical viewpoint, we now have the ability to verify marine ice sheet models, albeit that certain effects, such as buttressing, are not yet quantified/verified accurately, but new analytical tools are becoming available