Acute kidney injury (AKI) in deceased donors is not associated with graft failure (GF). We hypothesize that hemodynamic AKI (hAKI) comprises the majority of donor AKI and may explain this lack of association.In this ancillary analysis of the Deceased Donor Study, 428 donors with available charts were selected to identify those with and without AKI. AKI cases were classified as hAKI, intrinsic (iAKI), or mixed (mAKI) based on majority adjudication by three nephrologists. We evaluated the associations between AKI phenotypes and delayed graft function (DGF), 1-year eGFR and GF. We also evaluated differences in urine biomarkers among AKI phenotypes.2. Compared to non-AKI, donors with iAKI had higher odds DGF  and had lower 1-year eGFR [adjusted B coefficient (95% CI): -11  mL/min/1.73 m2]. hAKI and mAKI were not associated with DGF or 1-year eGFR. Rates of GF were not different among AKI phenotypes and non-AKI. Urine biomarkers such as NGAL, LFABP, MCP-1, YKL-40, cystatin-C and albumin were higher in iAKI.Of the 291 (68%) donors with AKI, 106 (36%) were adjudicated as hAKI, 84 (29%) as iAKI and 101 (35%) as mAKI. Of the 856 potential kidneys, 669 were transplanted with 32% developing DGF and 5% experiencing GF. Median 1-year eGFR was 53 (IQR: 41–70) ml/min/1.73miAKI was associated with higher DGF and lower 1-year eGFR but not with GF. Clinically phenotyped donor AKI is biologically different based on biomarkers and may help inform decisions regarding organ utilization. Less than 20% of patients on the waiting list receive kidney transplants each year, and approximately thirteen patients die every day awaiting a kidney transplant . DespiteMultiple studies have shown that deceased donor AKI is not associated with adverse recipient outcomes –12. LackS1A and S1B Fig) with the following abstracted donor variables: demographics , daily trends of hemodynamic status  renal function measures , medications , and microbiology . We securely distributed these profiles to three board-certified nephrologists, who independently reviewed AKI cases to adjudicate either as hAKI or iAKI. They were asked to use their clinical judgment to assess the phenotype of AKI based on the donor profiles as they would have done in routine clinical practice. All three nephrologists used common clinical markers such as serum creatinine trends, vital signs, volume status, vasopressor use, and presence of infection to accurately adjudicate the cases. They were blinded to the others’ adjudications, recipient outcomes and study urinary biomarker data. If a nephrologist could not confidently adjudicate hAKI or iAKI, they were asked to label the AKI as mixed subtype (mAKI). Final diagnosis was determined by majority adjudication. If all three nephrologists disagreed, the phenotype was designated as mAKI.This was an ancillary study from the Deceased Donor Study (DDS) and included 428 deceased donors with available charts from two organ procurement organizations (OPOs); Gift of Life Michigan and New York Organ Donor Network. Overall DDS methods have been described in detail elsewhere ,15. For After collection at time of organ procurement, urine samples were centrifuged at 1000×g for 10 minutes at 4°C, separated into 1 ml aliquots, and immediately stored at -80°C until biomarker measurement. The following urine biomarkers were measured: cystatin-C, albumin-to-creatinine ratio (UACR), interferon alpha (IFN), interleukin (IL-) 4, 6, 8, 10,18, kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (LFABP), neutrophil gelatinase associated lipocalin (NGAL), tumor necrosis factor alpha (TNF-α), chitinase-3-like 1 (YKL-40), epidermal growth factor (EGF), monocyte chemoattractant protein-1 (MCP-1), osteopontin (OPN) and uromodulin (UMOD). NGAL measurement was performed using the Architect platform (Abbott Diagnostics). LFABP was measured using latex-enhanced immunoturbidimetry with anti-human LFABP mouse monoclonal antibodies . All other urine biomarkers were measured using the Meso Scale Discovery platform , which uses electrochemiluminescence detection combined with patterned arrays.via chart review from the DDS cohort . The presence of moderate to severe ATI was also not different among the AKI phenotypes . There were no significant associations with DGF when comparing the hAKI and mAKI with non-AKI. Full multivariable model is shown in S2 Table.Out of 669 kidneys transplanted, 209 were from non-AKI donors, 184 were from donors with hAKI, 159 from donors with the mAKI and 117 from donors with iAKI. DGF occurred in 216 (32%) kidneys, with the highest rate of DGF in kidneys from donors with iAKI, 60 (51%), followed by hAKI, 59 (32%), mAKI, 44 (28%), and non-AKI, 53 (25%), p<0.0001. In univariable analyses, iAKI had significantly increased odds of DGF compared to non-AKI, but neither hAKI nor mAKI were significantly associated with DGF as shown in 2], compared to non-AKI [52  mL/min/1.73m2], mAKI [55  mL/min/1.73m2] and hAKI [57  mL/min/m2], p = 0.22} but did not reach statistical significance. On multivariable analysis, iAKI was independently associated with an 11 ml/min/1.73m2 decrease in eGFR compared to non-AKI [adjusted B coefficient (95% CI): -11.22 ] as shown in Fig 2. The full multivariable model is shown in S3 Table. DGF was not a significant effect modifier for the association between AKI phenotypes and 1-year eGFR.One-year eGFR was numerically lower in iAKI {iAKI  compared to non-AKI, but was not associated with DGF. mAKI was not associated with DGF or 1-year eGFR.We evaluated a subset of donors with persistent AKI at time of organ procurement, and out of 291 donors with AKI during hospitalization, 124 (43%) had persistent AKI at organ procurement. Of these, 16 (13%) had hAKI, 45 (36%) had mAKI and 63 (51%) had iAKI. Compared to non-AKI, iAKI was associated with nearly 3-fold odds of having DGF [2.92 ] and lower 1-year eGFR [-8.71  ml/min/1.73mWe evaluated associations between clinically adjudicated deceased-donor AKI and recipient outcomes in this multicenter study. We found that clinically phenotyped deceased-donor AKI had biological differences as evidenced by urine injury and repair biomarkers. We also found that donor iAKI happening earlier during donor hospitalization or ongoing at organ procurement was significantly associated with increased risk of DGF and lower 1-year eGFR but was not associated with early GF.Our study further explores the biological differences between hAKI and iAKI as identified by Barasch et al , and conAdditionally, clinically adjudicated iAKI was associated with increased risk for DGF and lower 1-year eGFR, further highlighting the potential benefit of accurately phenotyping AKI. hAKI is a functional change in the kidneys with reduction in filtration, but iAKI involves tubular cell injury and structural damage to the kidneys –27. GiveWhen evaluating GF, our study was limited by sample size but did not identify differing rates of GF among clinically adjudicated AKI phenotypes. Although future studies with larger sample size are needed to properly investigate the potential association between iAKI and GF, the findings of our study suggest that donor iAKI may lead to significantly lower 1-year eGFR but this decline in graft function may not be clinically meaningful to manifest as graft failure. These findings are consistent with our previously published data, which have shown that deceased-donor AKI, defined by terminal serum creatinine, is not associated with GF . This laOur findings need to be interpreted in the context of our study’s limitations. The three adjudicators may not be an accurate representation of the general physician population as all trained at the same institution. The phenotyping of AKI was mainly as nephrologists were encouraged to use their clinical judgment. However, this more accurately reflected real life clinical settings, as physicians rely on their clinical acumen to classify and phenotype AKI. Another limitation in our study involved our definition of AKI, which was based on a rise in serum creatinine, and did not account for potential creatinine level fluctuations in undiagnosed chronic kidney disease in the donors. In addition, the biomarker differences among phenotypes could have captured the clinical severity rather than the actual etiology of AKI as adjudicators assessed a wide variety of variables including laboratory, medication, as well as demographic data to adjudicate donor AKI cases. Histological confirmation of our clinically adjudicated AKI phenotypes was limited as ATI on biopsy was only found in <15% of kidneys in our study. This is limited by some practical concerns as procurement wedge biopsies are usually interpreted in a rush by non-renal pathologists, and hence tubular injury may not be accurately reported . HoweverIn conclusion, we have shown that clinically adjudicated deceased-donor hAKI and iAKI were biologically different by injury and repair urine biomarkers. iAKI was associated with higher rates of DGF and lower 1-year eGFR but was not associated with GF, whereas higher 1-year eGFR was noted for kidneys with hAKI at time of organ procurement. Clinically phenotyped deceased donor AKI may help inform decisions regarding organ allocation and utilization.S1 Figa: Example of a Deceased-Donor Profile . We created de-identified donor profiles abstracted donor clinical variables and distributed these profiles to nephrologists for adjudication. b: Example of a Deceased-Donor Profile . We created de-identified donor profiles abstracted donor clinical variables and distributed these profiles to nephrologists for adjudication.(TIF)Click here for additional data file.S1 TableAmong the adjudicated cases of hAKI, 51% had perfect agreement in the hAKI subtype, 17% had perfect agreement in the mAKI subtype and 32% had perfect agreement in the iAKI subtype.(TIF)Click here for additional data file.S2 TableThere were no significant associations with DGF when comparing the hAKI and mAKI with non-AKI.(TIF)Click here for additional data file.S3 Table2 decrease in eGFR compared to non-AKI.iAKI was independently associated with an 11 ml/min/1.73m(TIF)Click here for additional data file.


Acartia longiremis, Metridia pacifica, and Paraeuchaeta elongata remained active over most of the year. By contrast, the reproductive effort of Eucalanus bungii and Calanus marshallae was concentrated over the spring period and they spent most of the year in diapause as C5 copepodites. A delay in the timing of the spring bloom was associated with a shift in the phenology of all species. However, following the delay in spring bloom timing, recruitment to the G1 cohort was reduced only for E. bungii and C. marshallae. Recruitment successes of E. bungii and C. marshallae was also drastically reduced in 2010, an El Niño year, when spring temperatures were highest. Reasons for the observed differential response to spring environmental forcing, and its effect on upper trophic levels, are discussed.The temporal dynamics of five copepod species common to coastal waters of the Pacific Northwest were examined in relation to variability in spring temperature and phytoplankton dynamics in 2008, 2009, and 2010 in Rivers Inlet, British Columbia, Canada. The five species were differentiated by life history strategies.  Organisms have evolved diverse life history strategies to maximize their fitness in different environments . GeneralIn high latitude systems, the onset of the productive spring period occurs when an increase in stratification, due to the seasonal warming of surface waters and a decrease in wind speeds, shallows the mixed layer depth, allowing for an increase in phytoplankton growth rates . The timNeocalanus plumchrus, also point to an earlier biomass peak in association with warmer spring SST  to present the seasonal cycle of these species in the region; (2) to assess if the phenology of these copepods varied following interannual differences in spring bloom timing and spring temperature; (3) to determine how the interaction between overwintering strategy and spring bloom timing affects recruitment success. We focused our analysis on the spring season as juveniles of upper trophic levels that depend on zooplankton for successful recruitment appear during this period .M. Western Bounty across five sampling stations equally spaced along the length of Rivers Inlet , a deep (365 m maximum depth), 45 km long, on average 3 km wide fjord of glacial origin with a 137 m deep sill at its mouth on the Central Coast of British Columbia, Canada (a) were collected for the entire water column with a SBE 25 CTD. Profiles of temperature, salinity, and fluorescence were also taken every morning to a depth of 30 m from the Florence Daily site  were converted into chlorophyll a concentrations using filtered samples (see regressions in M. Western Bounty. Samples from the remaining stations were collected from the M. CCGS J. P. Tully using a 236 µm bongo net during a routine Cooperative Plankton Research Monitoring Program (COPRA) cruise of the Department of Fisheries and Oceans Canada.Detailed sampling design description can be found in , Canada . Prior tily site . The fluM. CCGS J. P. Tully using a 236 µm bongo net during routine COPRA cruises of the Department of Fisheries and Oceans Canada . Moreover, zooplankton abundance on a unit area basis (ind. m−2) was computed by multiplying the zooplankton density (ind. m−3) by the depth of the water column sampled.Detailed zooplankton taxonomic analysis design description can be found in s system , 2014, wAcartia longiremis copepodite stages C3 and below, M. pacifica copepodites stage C2 and below, and C. marshallae stage C1 copepodites are undersampled by the 150 μm mesh net  or C5 cohort  were back calculated using development times. No clear cohort could be distinguished for P. elongata and no birth date estimates were computed for this species. Copepod development time is temperature dependent and is generally modeled as a Belehrádek-type temperature dependent function   , we comp9–4.5 mm .a and α parameters to determine development times in Rivers Inlet for each depth layer/cruise/year/site/species/stage combination. The development time for the entire water column was then computed as an average weighted by the relative abundance of each stage and species in each layer. It was assumed that the zooplankton maintained the same vertical distribution between sites and years. For each cruise/year/site combination the weighted average by length of daylight of the day and night development times estimates was computed. Length of daylight was obtained from tables of standard times of solar rise/set for Port Hardy, British Columbia (127° 25′ W 50° 42′ N) downloaded from the National Research Council of Canada sunrise/sunset calculator . The estimated species and stage specific development times were comparable to development times reported in the literature   to chlorophyll concentration (mg m−3)  rather than EPR and chlorophyll. To estimate EPR from SEP, SEP estimates were multiplied by the ratio of the dry weight (DW) of Rivers Inlet M. pacifica adult females, 136 μg, and the M. pacifica egg DW, 0.62 μg. Egg DW was estimated from egg carbon weight derived from the egg diameter, 145 μm,  and numbers of egg produced to the back-calculated birthdate of the G(mg m−3) . The Eucfornicus . For Met 145 μm, , and eggg C μm−3 . Egg carg C μm−3  and finag C μm−3 . Egg pro0 of 2.7 .in situ chlorophyll concentrations as follows. Chlorophyll concentration depth profiles for each cruise/year/site/time of day combination were averaged over each of the vertical stratified zooplankton sampling layers and species specific EPR were obtained for each depth layer/cruise/year/site/time of day combination. The EPR of each layer was then multiplied by the abundance of adult females in each layer to estimates the number of egg produced. These estimates were summed to compute the total number of eggs produced over the entire water column. For each cruise/year/site combination the weighted average by length of daylight of the number of eggs computed during the day and night was computed. Length of daylight was obtained from tables of standard times of solar rise/set for Port Hardy. British Colombia (127° 25′ W 50° 42′ N) downloaded from the National Research Council of Canada sunrise/sunset calculator .The equations in Phytoplankton growth rates are fast and chlorophyll can fluctuate widely over the course of 2 weeks. Therefore, to better assess the seasonal fluctuations in EPR we also determined EPR from the daily chlorophyll time series. Daily chlorophyll depth profiles were only taken down to 30 m, thus EPR for the daily station is an estimate for the first 30 m of the water column. However, as chlorophyll below 30 m is very low , EPR dowParaeuchaeta elongata is a carnivorous copepod and as such it was felt that chlorophyll would not be an adequate proxy of food consumption and no EPR was computed for this species.Environmental parameters had been presented in detail elsewhere , 2014. BTemperature in 2010, an El Niño year, was consistently higher than both 2008 and 2009  and 3. DA. longiremis, which showed slightly longer development times in March of all years and in early April 2009 as compared to the rest of the sampling season  shifted between years. G1 abundance peaked in late April in 2008, but in early June in 2009  was observed from early April to late May in 2008 and 2010 and from early to late May in 2009 . By cont in 2009 . These s in 2009 . However in 2009 . Thus, tEucalanus bungii appeared to be dependent on the onset of the spring bloom for spawning initiation. Indeed, the late April peak in stage C1 E. bungii copepodites of 2008 and 2010 was a result of a spawning event in late March (1 C1 cohort occurred in early May at the onset of the 2009 spring bloom (E. bungii may rely on the onset of the spring bloom for spawning.te March , at the te March  and of ite March , and higte March . By contte March  as therete March , the estng bloom  and of hng bloom . The lacC. marshallae appeared to start reproduction prior to the bloom, but copepodites were recruited to the diapausing cohort only after the bloom. The observed early April stage C1 cohort in 2009 developed from eggs spawned in late March 2009 (1 C5 cohort developed instead from eggs produced in April in 2008 and 2010 and in early May in 2009 (By contrast, rch 2009 . Similarrch 2009 . The obs in 2009  at the o in 2009 .Acartia longiremis behaved as an income breeder, with the entire G1 population developing from eggs spawned during the bloom. When the bloom was a month later (1 cohort occurred a month later (th later , the appth later . Given tth later  resultedth later .Phenology of all the copepod species under study was delayed when the spring bloom occurred later. Thus, even the seasonality of omnivorous and carnivorous Type II copepods must be indirectly linked to the seasonality in phytoplankton production. Indeed, even when the timing of seasonal succession in zooplankton community composition was delayed in years characterized by a later phytoplankton bloom, the same zooplankton community composition was observed in Rivers Inlet over the period 2008–2010 .Paraeuchaeta elongata, the phenology shifts only applied to its early life stages, with adult densities remaining constant over the spring period. P. antarctica reproduction increased in spring, possibly as a result of improved feeding conditions following the ontogenic ascent and reproduction of overwintering copepods. In the Skagerrak, P. norvegica consumes 6–14% of copepod population spring production (P. elongata C1 copepodites in early spring in Rivers Inlet is consistent with enhanced feeding conditions in late winter and early spring, when M. pacifica and later C. marshallae exited diapause and commenced reproduction.For oduction . The obsMetridia pacifica displayed the earliest cohort timing of any species. The appearance of the first annual cohort of stage C1 copepodites always occurred approximately 45 days prior to the bloom and its birthdate was consistently 3 months before the bloom. For a continuously reproducing copepod species, appearance of a distinct cohort may be a result of an increase in bottom-up driven rates, such as egg production, or of a decrease in top-down processes, such as predation mortality. We showed that, in all years, the second cohort of M. pacifica may have been an example of an increase in egg production rate following an increase in chlorophyll. However, the M. pacifica G1 cohort was spawned in winter, when egg production was at its lowest, suggesting that survival may have controlled the appearance of this cohort. Cohort development of M. lucens in the North Atlantic was negatively related with C. finmarchicus abundance through intraguild predation of C. finmarchicus on the early stages of M. lucens (M. pacifica G1 cohort occurred prior to the ontogenic migration of C. marshallae in every year, suggesting that a similar mechanism may have been at play in Rivers Inlet. Unlike P. elongata, M. pacifica is a surface spawner (C. marshallae females. A careful assessment of M. pacifica winter and early spring mortality rates, predation pressure, and feeding history in relation to the spring phytoplankton bloom should be undertaken to determine the potential mechanism of the observed M. pacifica phenological variation.. lucens . The bir spawner  and thusA. longiremis peak egg production rates, and resulted in a phenological shift in the appearance of its G1 cohort. Adult A. longiremis abundance peaked at the end of the spring bloom, when dinoflagellates dominated the phytoplankton community composition (Acartia spp. biomass peak following a later dinoflagellate phenology was also observed in the North Sea (see review by A. longiremis was comparable in 2008 and 2010, 2 years of differing spring temperatures, we conclude that in Rivers Inlet an earlier buildup of the population is more dependent on an earlier timing of the spring bloom, and the associated high egg production rates, than on temperature. However, Acartia spp. phenology in other regions of the North Atlantic and in the Mediterranean appears to be more strongly associated with SST than phytoplankton phenology (see review by Acartia species. Most Acartia species overwinter as resting eggs, whose hatching is temperature dependent (A. longiremis diapauses in low numbers as immature females (A later onset of the spring bloom was associated with a delay in position . A laterependent . By cont females .A. longiremis, the dependence of E. bungii on spring bloom initiation for spawning resulted in a phenology shift in the timing of G1 copepodites following a shift in spring bloom timing. However, while in 2008 and 2010 the egg production pattern coincided with the timing of high egg production and cohort timing, in 2009 it did not. The estimated egg numbers were actually highest in late March 2009 because of high female abundance. The discrepancy between egg production estimates and the timing of appearance of the C1 cohort, and the lack of C1 copepodites until early May 2009, suggests that egg mortality was extremely high or that females were immature pre-bloom. In the Oyashio region significant numbers of mature E. bungii females only appeared at the onset of the spring bloom (E. bungii females may indeed have been immature prior to the bloom.Similarly to ng bloom , suggestE. bungii cohort timing may also have resulted from a later emergence from diapause. Indeed, a larger fraction of the population comprised adult females in February 2008 as compared to 2009, suggesting that moulting from C5 to adults occurred earlier in 2008 than 2009. The cue to exit diapause and initiate moulting in E. bungii is unknown. However, temperature has been proposed as an environmental control of diapause emergence and maturation in other copepod species (A shift in  species . Thus, tC. marshallae appears to be driven by differences in survival rather than changes in egg production. Reproduction started before the bloom, but recruitment was successful only for the cohort that developed into early copepodites during the spring bloom. These results are in agreement with observations from the Norwegian Sea demonstrating that C. finmarchicus copepodites do not develop beyond the early copepodites stages until the spring bloom (C. marshallae C1 copepodites appearing at the onset of the bloom (C. marshallae phenology appears to be controlled by differential survival of early copepodites rather than production and is dependent on the timing of the spring bloom.Phenology of ng bloom , and withe bloom . Thus, iE. bungii, a delay in phenology of C. marshallae may also have been driven by interannual changes in temperature. However, the timing of the terminal molt of C. marshallae was not later when water temperatures were colder in 2009; the fraction of adult females relative to stage C5 copepodites was actually higher in February 2009 as compared to February 2008. Therefore, internal controls of timing of diapause emergence such as accumulated lipid reserves may be more important than direct temperature control in determining the timing of emergence of C. marshallae C5 copepodites, as was concluded by C. finmarchicus. This suggests that temperature was not a direct driver the observed variation in C. marshallae phenology. Nevertheless, these results are in disagreement with data from the North Sea (see review by finmachicus phenology than phytoplankton bloom timing. This may be due to site-specific differences in the seasonality of phytoplankton dynamics, the seasonality of predation pressure, or the relative importance of advection as a driver of population dynamics. In Rivers Inlet, advection was not the main driver of C. marshallae population dynamics as compared to local demographic processes (Similarly to rocesses .E. bungii and C. marshallae following a delay in spring bloom timing has also been observed elsewhere (1 cohort size at high temperatures, during the El Niño year. It appears that continuous reproduction and an omnivorous feeding strategy allows Type II copepods to exploit seasonally variable food resources and to build their populations to a comparable size during years of varying spring environmental forcing and spring bloom timing. By contrast, species that focus their reproductive effort over the spring period appear more vulnerable to interannual variation in spring forcing.In terms of recruitment success, the response of the copepods under study was specific to each type of overwintering strategy. While the phenology of all copepod species was delayed following a later bloom, a delay in bloom timing was only associated with poorer recruitment success of Type I copepods. Reduction in recruitment of lsewhere . FurtherE. bungii and M. pacifica are subarctic oceanic copepods and C. marshallae and A. longiremis are boreal shelf copepods (P. elongata has generally not been associated with either Northern or Southern copepods. Our results are the first to demonstrate that further changes in community composition may be superimposed over the observed transport-driven variation following shifts in environmental forcing. This pattern may be particularly important for northern regions, which are presumably sources of these copepods, as well as for coastal embayments, such as fjords or inland seas, whose zooplankton community may not be as impacted by the circulation over the shelf break.Differential response of copepods to environmental forcing has long been recognized in the Northern California Current system, where Northern copepods dominate over Southern types in relation to ocean transport . Howevercopepods . P. elonE. bungii and C. marshallae copepodites (E. bungii and C. marshallae may have been lower when the bloom was later because of higher cumulative mortality of adult females from the time of diapause emergence to the time of successful copepodite recruitment during the bloom. Higher mortality of copepodites with a later birthdate, when temperature was higher, may also explain the lower recruitment when the bloom was late. The substantial decrease in Type I recruitment during the El Niño year provide further support for the hypothesis of higher mortality of Type I copepods at higher temperatures. The number of invertebrate predators increases over the season (E. bungii and C. marshallae.While it was not an aim of this study to assess the mechanisms for the observed variation in recruitment, some generalizations on the potential drivers of the observed pattern can be made. The 2009 spring bloom was less prolonged. Earlier termination of the bloom may have reduced growth rates of both epodites , and havepodites . Thus, oe season , predatie season , suggeste season , an asseE. bungii and C. marshallae had a second major spawning event in late summer/early fall. It remains to be assessed how phenology of fall spawning events changes interannually and how important fall spawning events are to recruitment in the following spring. If the successful recruitment window of Type I copepods in spring, marked by high chlorophyll concentrations and low mortality, will be reduced under future climate change scenarios, fall recruitment events may become essential to the maintenance of the annual reproductive success of these species.Both Since recruitment success of fish larvae relies on synchronization with peaks of their zooplankton prey , the obs10.7717/peerj.12238/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12238/supp-2Supplemental Information 2The equation is presented in Table 2.Click here for additional data file.10.7717/peerj.12238/supp-3Supplemental Information 3Acartia longiremis, CM = Calanus marshallae, EB = Eucalanus bungii, MP = Metridia pacifica.Error bars represent standard errors. AL = Click here for additional data file.


This study aimed to evaluate systemic thrombolysis experiences with recombinant tissue plasminogen activator (rtPA).Retrospective data were collected from 13 Turkish pediatric hematology centers. The dose and duration of rtPA treatment, concomitant anticoagulant treatment, complete clot resolution (CCR), partial clot resolution (PCR), and bleeding complications were evaluated. Low-dose (LD) rtPA treatment was defined as 0.01-0.06 mg/kg/h and high-dose (HD) rtPA as 0.1-0.5 mg/kg/h.Between 2005 and 2019, 55 thrombotic episodes of 54 pediatric patients with a median age of 5 years (range: 1 day to 17.75 years) were evaluated. These patients had intracardiac thrombosis (n=16), deep vein thrombosis (DVT) (n=15), non-stroke arterial thrombosis (n=14), pulmonary thromboembolism (PE) (n=6), and stroke (n=4). The duration from thrombus detection to rtPA initiation was a median of 12 h (range: 2-504 h) and it was significantly longer in cases of DVT and PE compared to stroke, non-stroke arterial thrombosis, and intracardiac thrombosis (p=0.024). In 63.6% of the episodes, heparin was initiated before rtPA treatment. LD and HD rtPA were administered in 22 and 33 of the episodes, respectively. Concomitant anticoagulation was used in 90% and 36% of the episodes with LD and HD rtPA, respectively (p=0.0001). Median total duration of LD and HD rtPA infusions was 30 h (range: 2-120 h) and 18 h (2-120 h), respectively (p=0.044). Non-fatal major and minor bleeding rates were 12.5% and 16.7% for LD and 3.2% and 25.8% for HD rtPA, respectively. At the end of the rtPA infusions, CCR and PCR were achieved in 32.7% and 49.0% of the episodes, respectively. The most successful site for thrombolysis was intracardiac thrombosis. HD versus LD rtPA administration was not correlated with CCR/PCR or bleeding (p>0.05).Systemic thrombolytic therapy may save lives and organs effectively if it is used at the right indications and the right times in children with high-risk thrombosis by experienced hematologists with close monitoring of recanalization and bleeding. The incidence of pediatric thrombosis is increasing due to advancements in neonatal and pediatric intensive care. Central venous catheters (CVCs), infections, surgery, immobility, trauma, congenital heart disease (CHD), vasculitis, underlying cancer, and thrombophilia are important risk factors for thrombosis ,2,3.EndoThis retrospective study was approved by the institutional ethics committee and approval was obtained from the patients’ legal guardians at admission to the hospital for the release of medical data for scientific purposes without including patient identity. A questionnaire form was designed and submitted to all Turkish pediatric hematology centers via electronic mail to collect the medical data of patients 0-18 years old who received systemic rtPA for thrombolysis. rtPA administrations for CVC clearance were not included in the study. Major surgery or central nervous system bleeding within 10 days and inability to maintain a platelet count of at least 50,000/µL with transfusion support or fibrinogen of at least 100 mg/dL were exclusion criteria. Concomitant heparin administration was at the decision of the attending physician. UFH dose of 5-10 U/kg/h and LMWH (enoxaparin) dose of 0.5 mg/kg twice daily were the prophylactic doses. Patients’ age, sex, underlying disease, location of the thrombus, period from thrombus detection to rtPA administration, previous anticoagulant treatments, initial and maximum rtPA doses, concomitant anticoagulant treatments, total duration of rtPA infusions, bleeding complications, thrombus resolution, amputations, organ loss, and outcomes after 3 months of anticoagulation were evaluated. Low-dose (LD) rtPA treatment was defined as 0.01-0.06 mg/kg/h and high-dose (HD) as 0.1-0.5 mg/kg/h . The cenDiagnosis of thrombosis and monitoring for clot resolution performed by color Doppler ultrasound examination, echocardiography, and/or computed tomographic angiography was acceptable. Complete clot resolution (CCR) was defined as objective radiologic imaging confirming recanalization of the occluded vessel and/or arterial thrombosis clinical examination showing equal pulse pressure, capillary return, and warm extremities. Partial clot resolution (PCR) was defined as objective radiologic imaging confirming partial recanalization of the occluded vessels. No resolution (NR) was defined as objective radiologic imaging confirming no change or thrombus extension in the occluded vessel.Centers reported performing laboratory monitoring by measuring prothrombin time, activated partial thromboplastin time, fibrinogen, anti-factor Xa, fibrin degradation products, D-dimer, platelet counts, and hemoglobin levels every 6-12 h. Patients were strictly monitored clinically for bleeding , any new emboli, allergic and anaphylactoid reactions, laryngeal edema, orolingual angioedema, rash, and urticaria. Centers also monitored blood pressure, heart rate, and respiration rate. If minor bleeding developed, the rtPA dose was either deescalated or rtPA was withdrawn transiently. They stopped the treatment and infused fresh frozen plasma (FFP) and/or red blood cells if major bleeding developed. If recanalization was seen in imaging studies, rtPA was stopped.For calculation of median and percentage, descriptive analysis was used; for comparison of two independent variables, the Mann-Whitney U test was used; and for comparison of more than two independent variables, Kruskal-Wallis H tests were used. Values of p<0.05 were defined as significant. All analyses were performed using SPSS version 13 .Thirteen pediatric hematology centers participated in the study. Questionnaire forms for patients aged 0-18 years who received systemic rtPA  between May 2005 and December 2019 were evaluated. The study included 55 thrombotic episodes of 54 patients  with a median age of 5 years (range: 1 day to 17.75 years). Two episodes (intracardiac and diffuse pulmonary thromboembolism) of a patient with CHAPLE syndrome were included. Eight of these cases were already published ,17. TablOut of 16 patients with intracardiac thrombus, five were preterm babies in the neonatal intensive care unit. They had umbilical catheters and ventilator support, and two of them also had septicemia. Five patients had CHD  and another two patients had cardiomyopathy. There were also patients with chronic pulmonary disease in the intensive care unit, acute lymphoblastic leukemia, lipin-1 deficiency , and CHAPLE syndrome (CD55 deficiency).Candida septicemia, hyperhomocysteinemia, and hyperlipidemia). There were also cases of Behçet’s disease, pyelonephritis, exposure to trauma during football activity, Down syndrome, epileptic seizures, idiopathic Budd-Chiari syndrome, and a patient taking oral contraceptive pills.Out of 15 patients with DVT, five had hematological diseases  and three had metabolic diseases  and one baby also had CHD. They also had umbilical catheters as a thrombus-provoking factor. CHD was also present in another four patients: Fallot tetralogy, ventricular septal defect (VSD) + patent foramen ovale + pulmonary hypertension, a newborn with aortic stenosis who underwent cardiac catheterization, and an infant with VSD who underwent transcatheter repair. Other patients had Kawasaki syndrome with coronary artery thrombus and pulmonary stenosis (n=2), while there was also an infant with bronchiolitis in the pediatric intensive care unit and an adolescent in the postpartum period with inherited homozygous antithrombin (AT) deficiency.In cases of pulmonary thromboembolism, underlying diseases were nephrotic syndrome, immune dysregulation , Behçet’s disease, multiple sclerosis with corticosteroid treatment, CHAPLE syndrome (CD55 deficiency), and obesity and pregnancy in a 17-year-old patient.Underlying diseases of the patients with stroke were hematological disease in two , infection in one (acute mastoiditis), and idiopathic thrombus in a toddler.As provoking factors for thrombus, there were CVCs, infections, and malignancy in 20 (36.4%), 10 (18.2%), and 7 (12.7%) of the thrombotic episodes, respectively. There was more than one thrombotic risk factor for some patients .In 63.6% (35/55) of the episodes, heparin was initiated before rtPA treatment. LMWH was preferred in 33 episodes and UFH in only two episodes. Six patients received FFP to replace plasminogen before administration of rtPA; two of them were infants. The period from thrombus detection to rtPA initiation was a median of 12 h (2-504 h); this period was significantly longer for DVT and pulmonary thromboembolism compared to stroke, non-stroke arterial, and intracardiac thrombi (p=0.024). When all 55 episodes were evaluated, no correlation was found between this period and thrombus resolution (p=0.75).There was a wide range of administered rtPA doses and durations. In 14 episodes, dose escalation, in four episodes dose de-escalation, and in two episodes temporary withdrawal of rtPA due to bleeding was applied. The median initial and median maximum rtPA doses were both 0.1 mg/kg/h (0.01-0.5). The total duration of rtPA infusions was a median of 18 h (2-120 h) and it was significantly shorter for cardiac thrombosis compared to pulmonary and arterial thrombosis (p=0.015).In this study, 33 patients received HD rtPA. Only one center used a standard rtPA treatment protocol. They used HD rtPA for 6 h and repeated infusion after 24 h if thrombus resolution was not achieved. They also transfused FFP at 10-20 mL/kg concomitantly. They did not use concomitant heparin. This center had five patients in this study. For four patients, they had to repeat rtPA infusions 3-6 times  and reported no bleeding complications. One of their patients with femoral and popliteal artery thrombus had CCR after only 6 h of rtPA infusion. Out of their five patients , two achieved CCR. Eight more patients from other centers received HD rtPA for only 2-6 h. When all patients receiving HD rtPA for ≤6 h  were evaluated, 2/9 achieved CCR with two minor bleeding complications. Patients receiving HD rtPA for 6 h at a time with or without repeated daily doses had two minor bleeding complications (2/13) and only two had CCR (2/13).In Concomitant heparin was administered in 58.2% (32/55) of the thrombotic episodes (10 UFH and 22 LMWH), in 24 episodes at the therapeutic dose and in eight episodes at the prophylactic dose. Therapeutic-dose heparin was preferred in patients receiving LD rtPA (p<0.05). In intracardiac thrombosis, concomitant anticoagulant administration was less frequent compared to stroke or DVT (p=0.046). In 18 episodes of this study, CCR was achieved, and in 11 of those, concomitant heparin was not used. In 10 of these episodes, the thrombi were intracardiac.Four major non-fatal bleedings (7.2%) developed; three were in patients with pulmonary thromboembolism  and one in a patient with coronary artery thrombosis (hematuria). Underlying diseases in the patients developing major bleeding were Kawasaki syndrome, Behçet’s disease, Evans syndrome, and multiple sclerosis; they all received concomitant heparin and three of them received LD rtPA for 18-48 h. FFP and packed red cells or only FFP or fibrinogen concentrate were administered as supportive treatment in major bleeding episodes. Antifibrinolytics were not used for any patient. There were minor bleedings in 12 episodes (21.8%) that could be easily controlled; these were epistaxis, gingival bleeding, venipuncture site bleeding, CVC exit site or incision site bleeding, subcutaneous hematoma, and bone marrow puncture site bleeding. When patients with and without rtPA-related bleedings were compared, no relationship was found with age, gender, initial rtPA dose, maximum rtPA dose, site of thrombosis, total rtPA duration, or concomitant use of anticoagulants. In 22 patients receiving LD treatment, there were three major and four minor bleeding complications. In patients receiving LD continuous treatment, rtPA doses exceeding 2 mg/h were used in older children and adolescents. When major bleedings in patients receiving LD were evaluated, a dose above 2 mg/h was administered for only one adolescent with pulmonary thromboembolism. This patient received concomitant LMWH and had hematuria and injection site bleeding. Two other patients on LD treatment and with major bleeding also received concomitant LMWH.As shown in There were two patients with unilateral renal artery thrombosis. The patient with inherited AT deficiency presented with left renal artery thrombosis in the postpartum period and left kidney atrophy developed. A 3.5-year-old patient with preexistent right kidney atrophy presented with left renal artery thrombosis and achieved CCR. There was an 11-year-old patient with pyelonephritis associated with left renal vein thrombosis (RVT); rtPA treatment was started 7 days after the diagnosis and PCR was achieved. This patient also already had right kidney atrophy at presentation. Unilateral blindness developed in a patient after stroke; the etiology of the thrombus was otitis media and mastoiditis. Amputations were performed for three patients with arterial thrombosis of extremities or fingers after unsuccessful thrombolytic treatments. Cirrhosis developed in a patient with idiopathic Budd-Chiari syndrome; this patient underwent liver transplantation. Three patients died due to prematurity complications of septicemia + necrotizing enterocolitis + bronchopulmonary dysplasia and one transplanted patient with juvenile myelomonocytic leukemia died due to veno-occlusive disease. Anticoagulant treatments were continued with LMWH for a median of 3 months (1-6 months) in all surviving patients except three patients who received coumadin and rivaroxaban and one non-compliant patient. Acetylsalicylic acid was also used for seven patients with arterial, cardiac, and venous thrombosis. The patient with CHAPLE syndrome also received eculizumab. When the patients’ outcomes were evaluated after 3 months of anticoagulation, CCR was increased to 74.5% from 32.7%.In The lack of prospective trials for the treatment of pediatric thrombosis with rtPA is a serious dilemma for pediatricians. The Thrombolysis in Pediatric Stroke Study started in 2010 but was ended by the National Institutes of Health in 2013 due to lack of accrual . In the Tarango and Manco-Johnson  recommenSimilar to our study, LD rtPA was shown to be effective and safe in a group of patients including preterm neonates and children . In the In the present series, early CCR and PCR rates were between 0% and 62.5% and between 37.5% and 75.0%, respectively, for different thrombus locations, but thrombolysis was significantly more successful for intracardiac thrombi and pulmonary thromboembolism . Fortunately, after two endovascular thrombectomies following unsuccessful systemic rtPA administrations, and with 3 months of anticoagulation, CCR ranged between 57% and 93% for different thrombus locations.In renal artery thrombus, rtPA treatment resulted in CCR in one of two patients. rtPA was also recommended for bilateral RVT with evidence of renal impairment . In a stIn three of four patients with stroke, PCR was achieved with thrombolysis. In the patient with left median cerebral artery thrombosis, PCR was achieved with rtPA administration even 12 h after diagnosis. Low rates of bleeding complications were reported with rtPA in cases of pediatric ischemic stroke , and repFor a 12-year-old patient with Behçet’s disease, rtPA was life-saving. Symptoms of thrombosis in multiple sites had developed within 15 days and systemic vasculitis was diagnosed. This patient was classified among the patients with pulmonary thromboembolism in this study, but he also had thrombi in the right atrium, vena cava inferior, bilateral iliac veins, vena porta, and vena hepatica. Administration of rtPA at 0.3 mg/kg/h for 46 h and concomitant UFH led to CCR. In this patient, there was major bleeding, which was controlled with temporary drug withdrawal and transfusion. There were four major non-fatal bleeding episodes (7.2%) in 55 systemic rtPA administrations. In addition to blood components, tranexamic acid may be used for bleeding complications. The use of antifibrinolytic agents for treating symptomatic intracranial hemorrhage that develops as a complication of thrombolytic therapy was also suggested . AntifibIn this series, CCR was achieved in only 13.3% of DVT episodes. In a pediatric study of 26 cases, there was no CCR in DVT, whereas CCR was achieved for 81% of arterial thrombi . SuccessIn vitro studies show that rtPA has more rapid clot lysis activity and high affinity for fibrin, and it induces the binding of plasminogen to fibrin; due to these characteristics, it is recommended in newborns and children over other thrombolytics ,27. In tThere are limitations of this study due to its retrospective multicenter nature and the heterogeneity of the patient population. For some patients, thrombolysis was initiated 2 weeks after thrombus presentation. Participating centers generally had no standard protocols for thrombolysis. Only some centers used concomitant heparin, either in therapeutic or prophylactic doses. Imaging studies for diagnosis and monitoring were not standardized. Data on serum fibrinogen, fibrin degradation products, anti-factor Xa, prothrombin time, and activated partial thromboplastin time were not presented in this study. Thrombosis requiring thrombolysis is rare in children compared to adults and each center was already trying to develop its own experiences with thrombolysis during the 14-year study period. Prospective multicenter pediatric studies must be planned using standard protocols with the administration of continuous LD rtPA for 6-72 h and HD for not more than 6 h at a time.Systemic thrombolytic therapy with LD or HD rtPA may save lives and organs effectively if it is used for the right indications and at the right times in children with high-risk thrombosis. This therapy should be applied by experienced hematologists under close monitoring of recanalization and bleeding.


Water quality parameters such as salt content and various pH environments can alter the stability of gels as well as their rheological properties. Here, we investigated the effect of various concentrations of NaCl and different pH environments on the rheological properties of TEMPO-oxidised cellulose nanofibril (OCNF) and starch-based hydrogels. Addition of NaCl caused an increased stiffness of the OCNF:starch (1:1 wt%) blend gels, where salt played an important role in reducing the repulsive OCNF fibrillar interactions. The rheological properties of these hydrogels were unchanged at pH 5.0 to 9.0. However, at lower pH (4.0), the stiffness and viscosity of the OCNF and OCNF:starch gels appeared to increase due to proton-induced fibrillar interactions. In contrast, at higher pH (11.5), syneresis was observed due to the formation of denser and aggregated gel networks. Interactions as well as aggregation behaviour of these hydrogels were explored via ζ-potential measurements. Furthermore, the nanostructure of the OCNF gels was probed using small-angle X-ray scattering (SAXS), where the SAXS patterns showed an increase of slope in the low-q region with increasing salt concentration arising from aggregation due to the screening of the surface charge of the fibrils. Cellulose, a polysaccharide, is the most abundant natural polymer . It can Starch belongs to a similar polysaccharide family as cellulose and is also widely used in the food  and pharRheological measurements have been widely used to study the structure–property relationships between various polysaccharide-based hydrocolloids ,30,31,32Both OCNF and starch are anionic hydrocolloids with different charge densities; therefore, understanding the interactions between these two species during gelation is important to describe their rheological properties. The addition of counter-ions  can be utilised to screen the repulsive forces between the OCNF fibrils ,7 and stTEMPO oxidised cellulose nanofibrils (OCNF) ,5 with ~Starch  with amylose content ~34%  was purcTransmission electron microscopy  analysis was carried out to investigate the morphology of the OCNF at an operating voltage of 200 kV. A dilute OCNF suspension (0.025 wt%) was added on a Cu-grid (mesh size 300), then negatively stained using uranyl acetate  (2 wt%) for enhanced contrast during TEM measurements .w/w) LiBr  while pullulan standards  were prepared at 2 mg mL−1 and incubated at 80 °C in glass vials overnight. A Perkin-Elmer  Series 200 High-Performance Liquid Chromatography (HPLC) equipped with a refractive index detector (RI), autosampler (35 °C), and column heater (80 °C) was used for peak resolution by size exclusion. An isocratic mobile phase of DMSO with 0.5% LiBr (w/w) at a flow rate of 0.500 mL min−1 and a stationary phase involving a series of columns beginning with a guard column  and two analytical columns in series . Total run time was 60 min, and injection volume for each sample was 50 μL, calibration curves were generated using pullulan standards with peak molecular weights ranging from 342 to 708,000 Da and correlation coefficients of R2 = 0.9996 ± 0.0001 (Rh) was determined using Mark–Houwink parameters for pullulan in DMSO/LiBr solution at 80 °C with the following parameters: K = 2.424 × 10−4 dL g−1, α = 0.68, and the dn/dc value was 0.0853 mL g−1 .From the amplitude sweep curves c, the liη) with respect to the shear rate of the gel systems are presented. The OCNF (1 wt%) gels with various concentrations of NaCl possessed shear thinning properties. The addition of salt to the OCNF gels (0.25 wt% NaCl) induced a significant increase of η and shear dependency.+ ions compete with the starch for the bound water in the gel system, resulting in less retrogradation, as suggested in the literature  with the addition of salt was also calculated. Results are presented in q) compared to the pure OCNF, while 0.50 wt% of NaCl in the OCNF gels showed a clear divergence [S(q) > 1] in the low-q region, suggesting the presence of salt-induced attractive interactions among the fibrils. A considerable divergence of S(q) value for OCNF gels with 1 wt% NaCl was observed, which further points toward the formation of larger aggregates of these negatively charged fibrils due to stronger fibril attraction at higher ionic strengths.SAXS patterns for OCNF gels with higher salt concentration (1 wt%) showed an apparent increase in the scattered intensity as well as the change of slope (to q−1.2 to q−2 at low-q) clearly demonstrates the salt-induced interactions among the starch molecules. Moreover, the slope obtained for starch with salt (q−2) was even higher than that of OCNF with salt (q−1.5), suggesting a more pronounced level of interactions of Na+ and Cl− ions with the starch molecules, inducing stronger attractions among them, or even aggregation into a fractal network or sheet-like structures [The SAXS patterns of starch at various salt concentrations were also investigated, as presented in ructures . This coq region  gels did not show any pronounced difference, except for the scattering pattern of the OCNF gel obtained at pH 4.0 at low-q a. The dierns see b, while q region arising from larger structures forming due to the screening of the surface charge of the fibrils. This work provides an in-depth understanding of the OCNF:starch gel network, which has the potential to be utilised as a rheological modifier in food, personal, and health care products.In this study, NaCl and pH-induced gelation of OCNF and starch in water systems were investigated. A progressive increase in the storage modulus of OCNF-only hydrogels with increasing salt concentration demonstrated the increase in the stiffness of the gels due to the surface charge screening of the cellulose fibrils by the counter ions. Additionally, the change in repulsive interactions  to the attractive regime  corresponded to their low-frequency dependent G′ and tanδ = G′’/G′ < 1 values in rheology, suggesting physical gel-like behaviour. When starch is added, the stiffness and viscosity of OCNF:starch hydrogels were further enhanced by the addition of NaCl . Increased ionic strength within the gel network disrupts the double layer of the charged particles, allowing stronger interactions between cellulose fibrils and starch molecules. However, at higher ionic strength , the OCNF:starch gel strength appears to reduce due to the formation of larger aggregated gel networks (resulting in precipitation). The OCNF:starch hydrogels were found to be stable in a pH range from 5.0 to 9.0, although at lower pH (=4.0), proton-induced fibrillar interaction was suggested to form stiffer gels. The reduced magnitude of the ζ-potential values obtained after the addition of salt as well as lowering the pH level was suggested to increase these interactions, promoting the aggregation behaviour of these hydrogels. SAXS analysis of OCNF also suggested that adding salt increased attractive interactions as observed by the increase in the slope at the low-


The aim of the present study was to evaluate the in vivo bone response to an additively manufactured zirconia surface compared to osseointegration into titanium (Ti) surfaces. Scanning electron microscopy, confocal laser scanning microscopy, and electron spectroscopy for chemical analysis were performed to assess the surface characteristics of implant specimens. For the in vivo evaluation, eight Ti implants and eight 3D-printed zirconia implants were used. The surface of four Ti implants was sandblasted, large-grit, and acid-etched (Ti-SLA group), while those of the other four Ti implants were left untreated (Ti-turned group). The zirconia implants had no further surface modification. Implants were placed into the tibiae of four rabbits; two received the Ti-SLA and zirconia implants and the other two received Ti-turned and zirconia implants. The experimental animals were sacrificed after four weeks of surgery, and the undecalcified microscopic slides were prepared. The bone–implant interface was analyzed by histomorphometry to evaluate the bone response. The degree of surface roughness showed that Ti-SLA was the highest, followed by zirconia and Ti-turned surfaces. The 3D-printed zirconia surface showed similar bone-to-implant contact to the Ti-turned surface, and Ti-SLA had the most bone-to-implant contact. The additively manufactured zirconia implant surface is biocompatible with respect to osseointegration compared to the commercially pure Ti surface. Since its initial use in the clinical application of dental implants, over 40 years ago, titanium has been considered as a gold standard material in terms of mechanical strength, chemical inertness, and biocompatibility, and it has shown a high success rate ,2. Howev2) dental implants have been investigated over the last 20 years to explore whether a zirconia implant can overcome the disadvantages of the titanium implant [The properties of zirconia  technology . AdditivThe aim of this study was to evaluate the bone response of titanium (machined and treated surface) versus zirconia implants, which were printed using AM methods inserted in rabbit tibiae. Furthermore, the physicochemical properties of 3D-printed zirconia implants were also analyzed. The null hypothesis underlying this study was that 3D-printed zirconia implants showed no significant difference in osseointegration compared to titanium implants.With the lithography-based ceramic manufacturing process, zirconia implants  were designed and 3D-printed. The zirconia implant possesses a content of 3 mol % yttrium oxide . The macroscopic design of the titanium implants  was transferred to the experimental zirconia implants, which had the same shapes and dimensions (a standardized diameter of 3.8 mm and a length of 10 mm).The implants’ surface was photographed by field emission-scanning electron microscopy . The element composition of each implant was performed by electron spectroscopy for the chemical analysis . Surface parameters for the surface topography of the implants were measured by confocal laser scanning microscopy . Each implant was analyzed at three different selected areas , which were averaged and assigned to the representative value for one sample . (1) FoA total of sixteen screw-shaped implants were used in this study. Eight zirconia implants  were applied as they were, without any surface modification. Eight titanium implants  were prepared and made of commercially pure titanium with/without surface modification . Four female New Zealand white rabbits  were included in this study. This investigation was approved by the institutional animal research ethics committee of Cronex (CRONEX-IACUC: 202004001) and were conducted following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines [At 4 weeks after implant installation, four rabbits were anesthetized and sacrificed by an intravenous overdose of potassium chloride for histologic assessment. The implants surrounding tissues and bones were surgically harvested en bloc. Each section was fixed in 10% neutral buffered formalin  for 2 weeks, which was followed by dehydration with a graded series of ethanol, and then they were embedded in methyl methacrylate . The embedded blocks were sliced using an EXAKT cutting unit , following the methods described in previous studies ,22,23, ap > 0.05). Descriptive statistics are described using the mean and standard deviation. To analyze the difference in the surface of implants, the Ti-turned, Ti-SLA, and zirconia implants were compared using the analysis of variance (ANOVA). To assess the difference in BIC and BA values between Ti-turned, Ti-SLA, and zirconia implant, the paired t test was applied. The statistical software R  was used for the analysis. Statistical significance was set at p < 0.05. Most of the outcome variables for data normalization were accepted using the Shapiro–Wilk test . There was no significant difference between the Ti-SLA and zirconia implants for Sa (p = 0.084). The means for Sdr were 165.22% (16.39%) for Ti-SLA, 23.01% (6.49%) for Ti-turned, and 106.93% (6.32%) for zirconia. The mean Sdr values showed that Ti-SLA was the highest, which was followed by zirconia and Ti-turned surfaces. The mean Sdr value for one surface was significantly different from that for another (p < 0.05).The results of ESCA analysis are shown in p = 0.012), but not in BA (p > 0.05). There were no significant differences between Ti-turned and zirconia implants in either BIC or BA (p > 0.05).The mature bone and newly formed bone might be distinguished by the Masson’s trichrome staining method . In the The topography and chemistry of implant in the bone are closely related to the reaction of the tissues surrounding the implants ,27. The Bone-to-implant contact (BIC%) and peri-implant bone area (BA%) are two important variables in quantitative analysis when evaluating the degree of osseointegration and discriminating between implant designs, surface composition, or surface modification ,29. In tThe implant surface modification enhances bone integration, which, in animal studies, is observed as higher BIC ,33. SennThe zirconia implants used in previous studies were usually manufactured by the conventional subtractive method, milling out of zirconia blanks. However, this subtractive manufacturing method leads to several problems, including the expensive molds and severe wear of cutting tools, limited reproduction of surface geometry, as caused by the predetermined size and shape of the milling instruments, and the axis of the computer numerical controlled (CNC) machine with a confined range of operation. Flaws such as cracking might also be created during the manufacturing process ,39,40,41The time of 4 weeks determined in this study was based on the previous literature ,44, whicAdditively manufactured zirconia ceramic implants are biocompatible at the level of commercially pure titanium implants, which have been reported to be successful for long-term clinical service. However, this study showed that the 3D-printed surface of these zirconia implants was inferior to that of the SLA titanium implants that are globally used in clinics for faster and stronger osseointegration. Considering the results of this in vivo study, an adequate modification method needs to be developed for the clinical application of these 3D-printed zirconia implants.


Nelumbo nucifera Gaertn.) starch granules slightly, leading to some detectable cavities or holes near hilum, weaker birefringence and granule agglomeration, accompanied with modified XRD pattern from C- to A-type starch and lower relative crystallinity, particularly for high moisture HMT modification. In contrast, annealing (ANN) showed less impact on granule morphology, XRD pattern and relative crystallinity. All hydrothermal treatment decreased the resistant starch (from about 27.7–35.4% to 2.7–20%), increased the damage starch (from about 0.5–1.6% to 2.4–23.6%) and modified the functional and pasting properties of lotus rhizome starch pronouncedly. An increase in gelatinization temperature but a decrease in transition enthalpy occurred after hydrothermal modification, particularly for hydrothermal modification involved with HMT. HMT-modified starch also showed higher pasting temperature, less pronounced peak viscosity, leading to less significant thixotropic behavior and retrogradation during pasting-gelation process. However, single ANN treatment imparts a higher tendency of retrogradation as compared to native starch. For dual hydrothermally modified samples, the functional properties generally resembled to the behavior of single HMT-modified samples, indicating the pre- or post-ANN modification had less impact on the properties HMT modified lotus rhizome starch.Heat-moisture treatment (HMT) changed the morphology and the degree of molecular ordering in lotus rhizome ( Starch is a semi-crystalline biopolymer that serves as a storage carbohydrate for energy reserves in many plants, including cereals, roots, tubers, seeds, and fruits. Starch granules in storage tissues can vary in shape, size, amylose/amylopectin ratio and functional properties . ApplicaNelumbo nucifera Gaertn.) is an important economic plant widely cultivated in China, India, Japan and Australia [Lotus rhizome  technique in a nondestructive way. Relative crystallinity (RC) can therefore be calculated by the ratio of the integrated diffraction intensity of the crystalline part to that of the amorphous part. X-ray diffraction (XRD) spectra and relative crystallinity (RC) of native/modified lotus rhizome starches were shown in After HMT, the peak 2θ at 5.6°  disappeared and the single peak at 17° split into double peaks at 17° and 18° , implying the crystalline type of HMT-modified starch transformed from C-type to A-type due to HMT modification. Gunaratne and Hoover  suggesteNevertheless, ANN did not change the XRD pattern and RC of lotus rhizome starches significantly, which is consistent with the findings from other studies on ANN-modified corn and acorn starches ,28. AlthDamage starch refers to the small starch fragments or particles broken up from the main starch granules. It may genetically come from starch itself or mechanical disruption during processing such as milling. During dough preparation, appropriate level of damage starch could improve the water absorption and dough mixing properties of flour ,30. The The amylose content of native lotus rhizome starches was about 18.38% and 16.43%, respectively . The amyBased on the rate of glucose released by amylase and its absorption in the gastrointestinal tract, Englyst, Kingman, and Cummings  classifiThe amylase digestion rates of starch are related to a barrier that slows down or prevents access or binding of enzyme to starch, and can be linked to the starch structural features that slow down or hamper amylase action as well . GunaratTheoretically, crystalline perfection and amylose-amylose and/or amylose-amylopectin interactions after ANN may increase the RS level, but this was not in agreement with the results in this study. It was suggested that ANN resulted in the slight irreversible swelling of granules, which may account for leaching of some amylose molecules out of the granules, formation of cracks on the granule surface; this change may negate the effect of crystalline perfection and starch chain interactions on enzyme susceptibility ,19,31. Ao, peak temperature Tp and conclude temperature Tc) represent the crystalline stability of starch granules, whereas enthalpy of gelatinization (∆H) reflects the melting of amylopectin crystal fraction with potential contributions from both crystal packing and helix melting enthalpies [o, Tp, Tc and Tc-To, but a decrease in ∆H, occurred after hydrothermal modification, particularly for hydrothermal modification involved with HMT. The high thermal energy imparted to starch chains by HMT would increase the flexibility of smaller fractions within starch granules, such as the amylose chain fractions and spacers (the segments that link amylopectin double helices to the backbone), and facilitate adjacent double helices interaction via hydrogen bonding. These interactions therefore result in more compact structure and restricted starch chain flexibility in bulk and also in inter-crystalline amorphous regions during granular swelling. Consequently, the enhanced crystalline stability by HMT would require a higher input of thermal energy to incur swelling and the disruption of the crystalline domain during gelatinization (higher Tc-To) [o also implies that HMT may partially break the relatively weak crystalline domain of starch granules in advance, thus decreasing the ∆H and RC . As shown in thalpies ,42. As sthalpies . An incro, Tp and Tc, although to a lesser extent than HMT-modification. However, the gelatinization temperature range (Tc-To) was lower. This is attributed to a possible increase in the amount of ordered double-helices, crystalline perfection, amylose–amylopectin or amylose–amylose interactions, amylose/amylopectin–lipid complex formation, and organized crystalline regions by ANN [ANN-modified starches also showed higher Ts by ANN ,45. The s by ANN ,28, indis by ANN . ThermalStarch applications are ultimately dependent on pasting viscosity. The pasting parameters of native/modified PF and WS starch measured by RVA are presented in Two common varieties of lotus rhizome harvested in Taiwan, namely Tsai-ou  and Shih-lian (abbreviated hereafter as WS due to its slightly whitish color and slender appearance), were purchased from a local farmer in Baihe district of Tainan, Taiwan.The native starch of lotus rhizomes was essentially isolated according to the method described by Rahman, Wheatley, and Rakshit . Figure w/v), was allowed to equilibrate overnight at 4 °C in sealed containers and incubated at 50 °C for 24 h. After cooling to room temperature, the samples were hot air-dried at 40 °C until the moisture content was less than 10% (d.b.). The annealed starch sample is hereafter referred to as ANN.Annealing treatment (ANN) was essentially performed based on the method reported by Liu et al. . Starch Heat-moisture treatment (HMT) was essentially carried out using the method described by Liu et al. . The moiDual hydrothermal modification of lotus rhizome starch was performed by applying HMT20 and ANN treatment in different sequences. For the HMT20 + ANN sample, lotus rhizome starch was subjected to HMT20 treatment (as described in Standard methods of AOAC  were ado2 + 1.28 mg/mL KI) was mixed with an appropriate amount of starch powder on the microscope slide and covered with a coverslip. The morphology and cross birefringence of the starch granules were viewed using an Olympus BX41 light microscope equipped with a simple polarizing attachment  and CCD camera .A drop of iodine solution  then examined using a scanning electron microscope system  under 3 kV.−1 and a step size of 0.02°. The relative crystallinity was quantified as the ratio of the crystalline area to the total area between 3° and 50° (2θ) using the PeakFit software .Crystallinity of native and hydrothermally modified starch samples was analyzed using a Powder X-ray diffractometer  operating at 45 kV and 40 mA. Starch sample was equilibrated over a saturated NaCl solution at room temperature (RH = 75%) for one week before analysis . The XRDDamage starch and amylose content were determined according to the AACC method 76-31.01  using a The content of resistant starch (RS) of native and hydrothermally modified starch samples were determined according to the analysis procedure provided by the Resistant Starch Assay Kit .The thermal properties of native and hydrothermally modified starches were measured using differential scanning calorimetry  according to the method of Man et al. [A Rapid Visco Analyzer RVA-Ezi  was used to determine the pasting properties of native and hydrothermally modified starches. The starch sample  was weighed into the RVA canister and water was added to make a total sample weight of 28 g. The following heating/cooling profiles were used: holding at 50 °C for 1 min, heating to 95 °C in 3.5 min, holding at 95 °C for 3 min, subsequently cooling to 50 °C in 3.5 min, and holding at 50 °C for 3 min. A constant paddle rotating speed (160 rpm) was used throughout the whole experiment except for a speed of 960 rpm for the first 10 s to disperse the sample. Viscosity profiles of lotus rhizome starches during the heating/cooling procedure were recorded.p < 0.05).All experiments were conducted in triplicate, and data are expressed as the means ± standard deviation. SPSS software  was used to carry out the one-way analysis of variance (ANOVA), and the post hoc analysis applied was Duncan’s test. Significant differences between means were determined at a confidence interval of 95% (Among the two lotus rhizome varieties studied, the functional properties of WS lotus rhizome starches were less affected by modifications than the PF lotus rhizome starch. As compared to ANN, HMT and dual hydrothermal modifications changed the starch properties to a greater degree. This suggests that crystallite disruption in HMT starch has a greater impact on starch properties than crystalline perfection in ANN starch. The results showed that HMT and dual modification were more effective in increasing thermal/shearing stability and decreasing the extent of setback, implying suitability for canned, baked and frozen foods. Although the ANN-modified sample did not show pronounced improvement in thermal/shearing stability, it had an adequate damage starch content, possessing the potential to be a flour treatment agent for the purpose of adjusting dough mixing properties. Moreover, hydrothermal modification techniques decrease the resistant starch content of lotus rhizome starch to varying degrees, and could be considered in order to adjust the digestibility of starch-based foods. This information could be useful for applications of lotus rhizome starch in various food preparations.


Oryza sativa L.) is a foremost staple food for approximately half the world’s population. The components of rice starch, amylose, and amylopectin are synthesized by a series of enzymes, which are responsible for rice starch properties and functionality, and then affect rice cooking and eating quality. Recently, proteomics technology has been applied to the establishment of the differentially expressed starch biosynthesis-related proteins and the identification of posttranslational modifications (PTMs) target starch biosynthesis proteins as well. It is necessary to summarize the recent studies in proteomics and PTMs in rice endosperm to deepen our understanding of starch biosynthesis protein expression and regulation, which will provide useful information to rice breeding programs and industrial starch applications. The review provides a comprehensive summary of proteins and PTMs involved in starch biosynthesis based on proteomic studies of rice developing seeds. Starch biosynthesis proteins in rice seeds were differentially expressed in the developing seeds at different developmental stages. All the proteins involving in starch biosynthesis were identified using proteomics methods. Most starch biosynthesis-related proteins are basically increased at 6–20 days after flowering (DAF) and decreased upon the high-temperature conditions. A total of 10, 14, 2, 17, and 7 starch biosynthesis related proteins were identified to be targeted by phosphorylation, lysine acetylation, succinylation, lysine 2-hydroxyisobutyrylation, and malonylation, respectively. The phosphoglucomutase is commonly targeted by five PTMs types. Research on the function of phosphorylation in multiple enzyme complex formation in endosperm starch biosynthesis is underway, while the functions of other PTMs in starch biosynthesis are necessary to be conducted in the near future.Rice ( Oryza sativa L.) is one of the most consumed cereal grains for half of the world’s population and ranks as the third-largest crop, after sugarcane and maize [O. sativa japonica and O. sativa indica [Rice  refer to the chemical modification events resulting from the covalent attachment of chemical groups, such as phosphate, acetyl, succinyl, methyl, and oligosaccharides, to amino acid side chains of the particular proteins . PTM is As the global population grows, it is expected that, by 2035, additional demand of approximately 112 million metric tons of rice needs to be produced to keep food security . UnderstThe enzymatic machinery for starch biosynthesis is summarized in 1 [2-B4 chains (DP ≥ 30) [In contrast, amylopectin, as compared with amylose, generally has a distinct fine structure and is synthesized by three main enzymes including SS, starch branching enzyme (BE), and starch debranching enzyme (DBE) . The SSs1 , and B2-DP ≥ 30) , respectBEs (EC 2.4.1.18) specifically introduce the branch point, the α-1,6-glucosidic linkage into the glucan chain by cutting the α-1,4-linked glucan and also transferring to another chain at the 6-hydroxyl position and thus is considered as a key enzyme regulating amylopectin structure . Three dDBEs (EC 3.2.1.10) are composed of two types, isoamylase  and pullulanase , and hydrolyze the improper α-1,6-glucans . Both ISBesides those main enzymes, starch phosphorylase  also plays a crucial role in starch biosynthesis and degradation ,52,53. POsGWD1 (Os06g0498400) is responsible for leaf starch excess1 (LSE1) [GWD1 in rice enhanced the phosphorylation at both C6 (9-fold) and C3 (2-fold) positions, increased amylose content, and displayed a minor change in starch granule morphology of endosperm [Phosphorylation is only known in vivo covalent modification of starch. Two important isoforms of glucan water dikinase (GWD), GWD1 (EC 2.7.9.4) and phospho glucan water dikinase  catalyze the phosphorylation at the C6 and C3 positions of glucosyl residues in amylopectin, respectively ,57,58,591 (LSE1) . The LSE1 (LSE1) . In addindosperm . Besidesndosperm ,65,66, sndosperm .OsSEX4 (LOC_Os03g01750/Os03g0107800)-knockdown rice caused an increase in starch accumulation in suspension-cultured cells, leaves, and rice straw, indicating that the function of OsSEX4 is conserved with Arabidopsis [Dephosphorylation is an essential mechanism required for starch degradation . Starch bidopsis . The trabidopsis .The disproportionating enzyme , a 4-α-glucanotransferase, cleaves the α-1,4 glucosidic bond, transfers the glucan moiety to the nonreducing end, and finally forms a new 1,4 glucosidic bond ,72,73. TThe initiation of starch granules is recently studied in order to understand the mechanisms and factors that influence the number of granules per plastid and the morphogenesis of granules . Severalosgbp mutants were significantly decreased in both leaves and grains, compared to wild type, leading to the reduction of starch content and number of starch granules with smaller size in leave and the presence of large chalkiness area in the endosperm [In rice, the functions of GBSS-binding protein (OsGBP) and Floury Endosperm6 (FLO6) are similar to PTST1 and PTST2, respectively. A newly identified OsGBP interacts directly with both GBSSI and GBSSII in yeast two-hybrid assay . The coindosperm .flo6 mutant showed many smaller granules with irregular shapes and rough surfaces [FLO6 plays an essential role in starch granule formation and contains N-terminal transit peptide and C-terminal CBM48 domain for plastid localization and binding to starch, respectively . FLO6 insurfaces .The proteome of each living cell refers to the entire proteins expressed by a genome, which is highly dynamic and altering in response to intra- and extracellular factors across time points . With diTo identify the tissue-specific expression in rice and understand the mechanisms that regulate starch biosynthesis, a total of 1022, 1350, and 877 unique proteins were identified from leaf, root, and seed tissues of rice, respectively, by using both 2-DE and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) coupled with multidimensional protein identification technology (MudPIT) . The uni2–4 chains elongation with the degree of polymerization (DP) ≥ 30 [Lin et al.  identifiDP) ≥ 30 . In contDP) ≥ 30  found SSIn cereal endosperm cells, starch granules are synthesized and increase in number and volume until maturity based upon the synergy of multiple enzymes ,30,84,85The identified starch biosynthesis-related proteins involved in rice endosperm development were summarized in The 396 differentially expressed protein spots were identified in Nipponbare during the early (6–8 DAF), mid (8–12), and late (12–20) stages of rice seed development . ISA I, Lee et al.  reportedBesides starch biosynthesis-related proteins, proteins involving in other metabolic processes such as glycolysis, TCA-cycle, lipid metabolism, and proteolysis were also detected at higher levels in the fully mature grain (desiccation phase), compared to the developing stages, suggesting that the accumulation of these proteins might be for seed germination .Zhang et al.  found thYu et al.  identifiOverall, the expression patterns of proteins involving in starch biosynthesis, starch degradation, and starch phosphorylation are particularly responsible for rice seed development. Most of those reported proteins are continuously increased during 6–20 DAF. However, proteins involving in starch debranching, degradation , and phosphorylation (GWD) are markedly decreased at 30 DAF and then increased to complete seed development.HT particularly affects the yield and quality of rice  but no sHT at 35/30 °C (day/night) decreased the GBSS abundance of japonica rice (TNG 67), leading to the lower amylose content observed at 15 DAF (12.3 ± 0.5%), compared with the control temperature at 30/25 °C (15.6 ± 0.4%), while those of indica rice (TN1) were relatively stable under both temperatures . AccordiCompared to the perfect grain, SSI, SSII, PUL, GBSSI, and BEIIb were downregulated, while AMY (AmyII-3) was increased in chalky grain in both moderate (24.4 °C) and HT (28.0 °C) conditions . DBE andMoreover, Timabud et al.  reportedAltogether, HT activates starch degradation rather than starch biosynthesis through the reduction of GBSSI, SSI, SSII, SBEIIb, DBE, and PUL, while some proteins, such as AGPase L, GBSSI, SBEI, and AMY are increased and contributed to the lower amylose content and the higher the chalkiness rate in rice endosperm.PTMs can alter structure formation, activity, stability, structure, and localization of proteins, which are necessary for cellular functions ,85. FiveProtein phosphorylation is a reversible process regulated by kinases and phosphatases and regarded as one of the most important PTMs . The stuRecently, the phosphoproteins involved in starch biosynthesis of indica rice cultivars (9311 and Guangluai4) including AGPase (three sites) AGPS2 (five sites), SSIIa (one site), SSIIIa (one site), BEI (four sites), BEIIb (two sites), PUL (three sites) and Pho1 (two sites) were reported by Pang et al.  Table 4Table 4. Interestingly, one phosphopeptide of both AGPase and SSIIIa showed consistency between japonica and indica rice ,31. AGPaTriticum aestivum), phosphorylation can activate SBEIIa and SBEIIb enzymes contributed to the protein complex forming of SBEIIa, SBEIIb, and Pho1 at 12–25 days after pollination (DAP) [In amyloplasts, starch biosynthesis isozymes have been demonstrated to display as a complex form (or protein–protein interactions), especially through the regulation of phosphorylation ,119,120.on (DAP) . On the on (DAP) . FurtherZea mays L.) endosperm, SSI, SSIIa, and SBEIIb form a trimeric complex in which SBEIIb is phosphorylated [amylose extender, ae− mutant) impacts the protein–protein interactions among SSI, SSIIa, and SBEIIb complex, which is formed in wild type [ae− mutant. Since the SBEIIb is replaced by SBEI, a reduction of branch points with longer glucan chains was observed in ae− mutant, as compared to the wild type [Hordeum vulgare), protein complex formation of SBEIIa, SBEIIb, SSIIa, and SSIIIa was increased by the presence of ATP [In maize  from acetyl–coenzyme A (CoA) is donated to Nε-terminal amine of a lysine residue through acetyltransferases and removed by deacetylases [Lysine acetylation, a highly conserved PTM in organisms, is well known for the regulation of transcription  and repoetylases . The aceetylases . In riceIn total, 2, 2, 2, 10, 2, and 8 sites of lysine acetylation modifications identified from starch granules of mature rice endosperm were reported for AGPase S2, AGPase L2, GBSSI, SBEI, SBEIIb, and Pho L, respectively  Table 5Table 5. Compared to root, leave, flower, and pollen, the prominent acetylated proteins of rice were observed in seeds at 7, 15, and 21 DAF, as well as in mature dry seeds . A totalKF (5), KY (4), KXXXR (3), KXXXXR (1), KXXXK (4) and KH (4)  .Brachypodium distachyon L. enhanced the activity in glycolysis and decreased the activity in gluconeogenesis [Lysine acetylation has a strong impact on the biochemical functions of proteins . For exaogenesis . On the ogenesis . It is pSuccinylation is recently identified as one of the PTMs on lysine residue ,137 and B. distachyon L. [Previous studies indicated that succinylation has a potential impact on protein structure and functions  as well chyon L. , patchouchyon L. , and teachyon L. . Howeverhib is a highly dynamic PTM found on both histone and nonhistone proteins affecting the histone–DNA association and playing role in diverse biological processes [hib introduces a huge change in size, as compared to lysine acetylation, and particularly forms hydrogen bonds with other molecules via its hydroxyl group [Krocesses ,145. Khimal, a lately identified lysine acylation, is evolutionarily conserved in mammalian and bacteria cells [mal triggers more dramatic structural changes than both lysine acetylation and methylation on the substrate proteins [hib and Kmal were identified from the developing rice seeds at 15 DAF reported by Meng et al. [In contrast, Kia cells ,147 and ia cells ,146. Kmag et al.  and Mujag et al. , respecthib proteins were reported in rice [hib were observed on SBEI (33 sites), followed by Pho L (32 sites) and AGPase L2 (21 sites). The Khib function on targeted proteins remains unknown in rice. However, there was evidence that Khib modification may introduce the conformational changes of Enolase1 and alter the substrate binding [hib increased the hydrophobic solvent-accessible surface area and decreased the enzymatic activity of UvSlt2 [A total of 2512 K in rice  and amon binding . Khib inf UvSlt2 .mal, seven starch biosynthesis proteins out of 247 malonylated proteins were reported [mal remain largely unknown. The malonylation has an important role in the enzymatic activity of various proteins. For example, the enzymatic activity of malonylated fructose bisphosphate aldolase B (ALDOB) was decreased by 20%, as compared to nonmalonylated ALDOB [mal had the impaired mitochondrial function and fatty acid oxidation [For Kreported . The numed ALDOB . Malonyled ALDOB . Moreovexidation .(1)Proteome alteration under climate change environment: Recently, the global population is facing challenging problems caused by global warming and climate change, which have a great impact on rice yield and quality. Further studies are needed to determine the consequences of climate change, e.g., high/low temperatures, carbon dioxide levels, drought stress, etc., on starch biosynthesis mechanism and regulation by using proteomic analysis.(2)hib showed the highest number of targeted starch biosynthesis proteins (17 proteins), while the lowest number was observed in succinylation (2 proteins). Whether more PTMs would be found under the specific genotype or under the specific abiotic conditions such as heat stress, high carbon dioxide levels, etc., is unknown.The number and new types of PTMs in rice seeds: Although five types of PTMs were identified from rice seeds, whether there are other PTMs in rice seed has not been fully addressed. For the number of PTMs sites, K(3)The roles and regulation mechanisms of PTMs on starch biosynthesis: Little is known about the roles of individual PTM on the starch biosynthesis proteins and the impact of PTMs on enzymes’ activities, protein–protein interaction (protein complex formation), and starch functionality. The phosphorylation is well reported in protein complex formation during the starch biosynthesis process in the endosperm of cereal crops. In-depth regulatory studies on protein–protein interactions are necessary to understand the role of protein complex formation in starch biosynthesis in different crops.Even though identification of the proteomics and PTMs has been conducted in rice developing seeds, there still remain significant gaps in their regulations in starch biosynthesis. This review presents the significant proteins associated with starch biosynthesis in rice seeds and their expression profiles through different developmental stages and high temperatures. Most of the key proteins in starch biosynthesis are generally increased in the endosperms during 6–20 DAF. SSIIIa and AMY are promising proteins in chalky formation. Hight temperature initiates starch degradation rather than starch biosynthesis and then also results in the reduction of amylose content as well as the increase in chalkiness in rice seeds. Twenty starch biosynthesis proteins are targeted by five types of PTMs including phosphorylation, lysine acetylation, succinylation, lysine 2-hydroxyisobutyrylation, and malonylation. PGM is commonly targeted by all the five PTMs types . Phospho


For more than 40 years, under the leadership of four editors and two publishers, The International Journal of Aging and Human Development (IJAHD) has featured multidisciplinary scholarship related to aging processes and older adults. With the publication of eight issues a year, with over 800 pages of scientific content, the IJAHD places emphasis upon psychological and social studies of aging and the aged. However, the Journal also publishes research that integrates observations from other disciplines that illuminate the "human" side of gerontology. A more recent focus includes midlife development, as well. About half (47%) of the publications in the IJAHD are from international colleagues. This presentation will discuss tips for both international and US-based scholars for ensuring timely reviews and positive decisions for manuscript submissions, including such areas as key words, suggesting unbiased reviewers, formatting, writing mechanics, clearly-articulated methods, and a sound theoretical basis.


For the last 9 years I have been the President of the Romanian Society of Ophthalmology, meaning 2 mandates and a little bit more, due to the pandemics.A long period of time, but a fulfilling one, from my point of view!The decisions were taken easier due to the collaboration, the permanent exchange of ideas, the fact that we know and we appreciate each other.Annual congresses with significant participation, reunited with SEEOS  and/ or BSOS , with well-known national and international speakers were organized. The conferences in Sinaia were very much appreciated by the colleagues and now we all hope to meet again there in 2022 and not on Zoom.Beside the paper works presented at the Romanian Society of Ophthalmology (RSO) Congresses, prize awarding contests for residents and nurses were organized. Previously, the participations were honorific but we changed this and offered significant money prizes and the results were seen. On the other hand, some companies offered scholarships in different European clinics and this was very much appreciated by the young doctors.The Society bought the educational “One network” programme from the American Academy of Ophthalmology for professional purposes, which was well received by the Romanian ophthalmologists.The Society was preoccupied by the national organization of the International Council of Ophthalmology exams, with significant reduced taxes because they were collected by RSO.A major decision with significant impact in our collectivity and international appreciation was the transformation of “Oftalmologia” journal into “Romanian Journal of Ophthalmology”, published in English. One of the results of this change was the fact that we received for publishing many articles from different parts of the world.The implementation of a new curricula for specialist doctor exam, requested by the University staff for the past few years, was obtained with great effort and in collaboration with the Ministry of Health.A new headquarter situated in the central part of Bucharest was bought for the Society, facilitating the collaboration with the doctors, medical companies, and authorities.It was a constant and fulfilling collaboration with the medical companies, companies that were involved financially. Without them, the Society’s activities would have been poorer. Their help made possible a series of online webinars, organized at high standards and their activity continues in the present and hopefully will also continue in the future.In conclusion, it must be said that the good, collegial, and responsible collaboration was a constant in the activity of RSO board and now we confidently look to the future leadership.With friendship and consideration,Assoc. Prof. Mircea Vasile Filip, MDRSO President 2012-2021