The differences in the levels of bone biochemical markers between patients and controls were analyzed by one-way ANOVA or chi-square test. The relationship between HbA1c and bone biochemical markers was analyzed by multivariate regression, forest plot and fitted curve.p < 0.001; 48.47 ± 27.27 vs 65.86 ± 21.06, p < 0.001, respectively), but the bone resorption markers β-crossLaps (β-CTX) was no difference between the two groups . Multivariate regression showed that HbA1c was inversely associated with N-MID osteocalcin and PINP after adjusting for age, BMI, menopause’s years, diabetic duration, TC, TG, HDL-c, LDL-c, creatinine, UA and eGFR. The adjusted coefficients for N-MID osteocalcin and PINP per 1% HbA1c decrease were − 0.71  and − 1.79 , respectively. A segmentation effect was seen in the fitted curve between HbA1c and β-CTX with an inflection point at 7.4% of HbA1c, the highest quartile of β-CTX (> = 0.74 ng/ml) showed a significantly negative with HbA1c. No significant association was seen between HbA1c and other biochemical markers.Bone formation markers including N-MID osteocalcin and procollagen type 1 amino-terminal pro-peptide (PINP) were decreased in postmenopausal women with T2DM compared to controls (17.42 ± 9.50 vs 23.67 ± 7.58, Our study found that bone formation was inhibited in postmenopausal women with T2DM, but bone resorption was not affected, and poor glycemic control was related to lower levels of bone formation, may increase the risk of bone fracture in postmenopausal women with T2DM. It can also lead to a dysregulation in the handling of calcium, phosphorus and magnesium, thus leading to a series of complications such as cardiovascular disease, peripheral vascular disease, retinal disease, and neuropathy. Bone is also affected by T2DM. Osteoporosis is a long-term complication of T2DM that is prevalent in postmenopausal women, and previous studies have shown that risk of fractures was significantly higher in patients with T2DM than in those without T2DM; therefore, researchers have focused on the effect of diabetes on bone mineral density (BMD). BMD has been shown to be decreased or normal in T2DM patients in some studies –4, whileHowever, studies on bone biochemical markers in T2DM have shown conflicting results, with the inhibition of bone formation being reported in most cases –9; a recThis was a cross-sectional study that included patients from the First Affiliated Hospital of Anhui Medical University diagnosed with T2DM. Patients with the following diseases that influenced the bone metabolism ware excluded: hyperparathyroidism, hypercortisolism, osteoporosis, chronic liver disease, malignant tumor, Cushing’s syndrome, acromegaly, rheumatoid arthritis, thyroid disease, ankylosing spondylitis, chronic obstructive pulmonary disease and chronic kidney disease including diabetic nephropathy. Patients who had a history of taking estrogen, parathyroid hormone, glucocorticoids (more than 3 months), anti-seizure drugs, bisphosphonates was also excluded. A group of 237 postmenopausal women were ultimately included in this study. The average age of the subjects was 64.41 ± 9.23 years. Recruitment occurred from January 2017 to October 2018, in additional, postmenopausal women visited the hospital only for a health examination matched for age to T2DM with normal glucose tolerance from healthy examination center of the First Affiliated Hospital of Anhui Medical University were enrolled in our survey, according to the medical examination report, 93 controls meet our standard in the end, average age of the controls was 64.61 ± 7.62 years. T2DM was defined according to the updated and approved diagnostic criteria for diabetes of the American Diabetes association (ADA) released in 2017, when fasting plasma glucose was (FPG) ≥7 .0mmol/L and/or 2-h posted-glucose (PG) was ≥200 mg/dL (11.1mmol/L) during oral glucose tolerance test (OGTT) and/or HbA1c ≥6.5%; normal glucose tolerance was a status that fasting plasma glucose was (FPG) < 6 .1mmol/L and/or 2-h posted-glucose (PG) was < 7 .8mmol/L) during oral glucose tolerance test (OGTT). This research was approved by the Ethics Review Committee of the First Affiliated Hospital of Anhui Medical University (Reference number Quick-PJ2018-11-11).3 and other variables, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-c), low-density lipoprotein-cholesterol (LDL-c), creatinine, uric acid (UA) and estimated glomerular filtration rate (eGFR).Blood samples were collected after fasting for at least 8 h. The samples were then centrifuged for 5 min at 3500 r/s within 2 h. The serum was then stored at − 20 °C and was subsequently used to measure the levels of bone biochemical markers, including β-CTX, PINP, PTH, N-MID osteocalcin and 25(OH)D3 were measured using electrochemiluminescence immunoassay , and glycosylated hemoglobin A1c (HbA1c) was measured by high-performance liquid chromatography (HPLC) using Bio-Rad D-10. Serum TC, TG, HDL-c, and LDL-c were detected by immunoturbidimetry. The within and between batch coefficients of variation were both < 8%. The laboratory was certified by ISO 15189.The bone markers PTH, PINP, β-CTX, N-MID osteocalcin and 25(OH)D2). The information containing patient’s age, BMI, menopause’s years, duration of T2DM, diabetic complications including microvascular complications and diabetic retinopathy (DR)) and macrovascular complications , history of fracture, use of stain medication, and treatment type of hyperglycemia were collected.The weight of the patients was measured with a calibrated balance-beam scale and height was measured using a stadiometer. Body Mass Index (BMI) was calculated using the formula weight divided by the square of height (kg/m2), diabetic duration (< 10 or ≥ 10 years), history of fracture (yes or no), microvascular complications (yes or no), macrovascular complications (yes or no), and treatment type , use of stain medication(yes or no), the results were presented through forest plot. Spline smoothing of the generalized additive models was performed to explore the relationship between HbA1c and these outcomes.All continuous variables were expressed as the mean ± standard deviation(SD) or median (interquartile range), while categorical variables were described as a frequency or percentage. One-way ANOVA was used to compare continuous variables, and a chi-square test was used for categorical variables. Multiple regression analysis was used to estimate the independent association between HbA1c and bone biochemical markers while adjusting for potential confounders. Stratified and interaction analyses were implemented according to age (< 60 or ≥ 60 years), menopause years (< 10 or ≥ 10 years), BMI  and the software Stata . P < 0.05 was considered statistically significant.All of the analyses were performed using the software R for statistical computing . Only 17 patients had a history of fracture; 183 patients were accompany with microvascular complications; 144 patients were suffered with macrovascular complications; 63 of 214 patients received insulin treatment only, 23 patients used anti-diabetic drugs only, 151 patients were using combination therapy(Table p < 0.05), but no significant difference in the level of serum PINP, β-CTX and 25(OH)D3 was observed. History of fracture, microvascular or macrovascula rcomplications and treatment strategy also had no difference between the HbA1c < 8.5% group and the HbA1c > 8.5% group, however, the use of statin medication was different obviously, moreover, individuals with higher HbA1c level were likely to have a higher levels of eGFR and TG but a lower levels of creatinine and HDL-c.A total of 214 postmenopausal patients with T2DM were enrolled in this research. The clinical information of the recruited patients was listed in Table To investigate whether the bone biochemical markers levels in T2DM are associated with HbA1c, we performed a multivariate regression analysis after adjusting for different variables Table . Linear ’s years (< 10 or ≥ 10 years), and BMI (< 24 or ≥ 24 kg/m2) diabetic duration(< 10 or ≥ 10 years), history of fracture(yes or no), microvascular complications (yes or no), macrovascular complications (yes or no), treatment type  and use of stain medication(yes or no) are shown in the forest plot .Stratified and interaction analyses according to age (< 60 or ≥ 60 years), menopauseAs for the linear relationship between HbA1c and N-MID osteocalcin, and between HbA1c and PINP, we speculated that a nonlinear relationship existed between HbA1c and the other biochemical markers studied. We used smoothing splines generated in the generalized additive models to explore HbA1c and these outcomes Fig. . A lineaClearly, glucose metabolism is closely related to bone metabolism, as bone biochemical markers in patients with diabetes were affected by many factors, such as the type of diabetes, the presence of complications, history of fractures, the treatment type and so on. However, how T2DM affects bone metabolism in postmenopausal women remains unclear. Studies on bone turnover markers in type 2 diabetic patients have reported conflicting results, and a consensus has not yet been reached. In this investigation, we explored the association between bone turnover and type 2 diabetes in postmenopausal women.In this research, we found that the bone formation markers N-MID osteocalcin and PINP were lower in postmenopausal women with T2DM than in the controls, in agreement with most studies –9. The iAt the same time, the bone resorption marker β-CTX did not show any discrepancies between diabetic patients and controls in our investigation. β-CTX was a confusing and controversial biochemical marker in T2DM all the time. Consistently with our results, β-CTX had no differ in T2DM patients and non-diabetic patients in some studies , 16, oth’t alter bone metabolism [Among postmenopausal women with T2DM, we found N-MID osteocalcin and PINP was negatively correlated with HbA1c in type 2 diabetes with or without adjustment, indicating that a poor glycemic control may be associated with a reduction of bone formation. After adjusting for the variables we collected, the opposite relationship was weakened, interaction analyses in stratified subgroup showed age, menopause years, BMI, diabetic duration, history of fracture, microvascular complication, macrovascular complication and treatment of T2DM did not influenced the relation between HbA1c and PINP. However a stratification analysis according to history of fracture(Yes or No) conducted showing a significant difference of N-MID osteocalcin in the two subgroups based on HbA1c, which means HbA1c does not independently affect the level of N-MID osteocalcin, but is affected by a history of fractures. Some previous reports detected insulin treatment may not affected the bone biochemical markers, metformin treatments could decrease the bone biochemical markers , 21; nevtabolism . A studytabolism , a popultabolism , the effIn a type 1 diabetic mouse model, Botolin et al.  detectedA higher level of HbA1c induces insulin secretion and hypoinsulinemia inhibits the process of bone formation, and PINP was directly associated with insulin resistance, indicating that HbA1c affects insulin resistance and insulin sensitivity. Reactive oxygen species (ROS) was increased in diabetes by a variety of mechanisms including increased AGEs . Therefo’ characteristics indicated in a systematic review [’s research may help us to interpret this phenomena, when HbA1c increased from 6.93 to 7.46%, DN progression was significantly accelerated in patients with T2DM, resulting in an increase in β-CTX exclusion [An interesting result in our study is the identification of a segmentation effect between HbA1c and β-CTX; an inverse relationship between HbA1c and β-CTX was found when HbA1c was less than 7.4%, but no association was noted when HbA1c was greater than 7.4%. When quartered the level of β-CTX, the highest quartile of β-CTX (> 0.74 ng/ml) showed a significantly negative with HbA1c. Recently, a study involving people with a normal glucose tolerance found that β-CTX was positively associated with HbA1c . Ogata, c review . Kaori Kxclusion . Besidesxclusion , and a tAlthough many confounders was adjusted in our research, there are also some limitations. First, our sample size was not large enough. Second, the cross-sectional survey used in this study limited our ability to identify etiological factors, and since our survey was focused on postmenopausal women suffering from T2DM, the findings herein reported may not suitable for men. Third, we were unable to obtain all of the information regarding all the subjects involved in this study including BMD and risk factors for fracture in order to explore their relationship with HbA1c, bone biochemical markers and BMD. Fourth, detail information regarding the anti-diabetic medications taken by the subjects was not available in this study, since it has been shown that the level of bone biochemical markers was also influenced by different kinds of hypoglycemic drugs.Bone formation was inhibited in postmenopausal women with T2DM, but no alteration of the bone resorption marker β-CTX was observed compared to controls. HbA1c was inversely related to N-MID osteocalcin and PINP. Therefore, a poor glycemic control may aggravate bone loss, increasing the risk of fracture in patients with T2DM.


Fifteen healthy older men (69 ± 4 years) attended two experiment sessions and consumed either 140 mL of concentrated beetroot juice (800 mg NO3−) or placebo (NO3−-depleted beetroot juice) in a randomised, double blind, cross-over design study. Plasma nitrite (NO2−) and NO3−, blood pressure (BP), augmentation index (AIx75), pulse wave velocity (PWV), FMD of the superficial femoral artery, and PLM hyperaemia were measured immediately before and 2.5 h after consuming NO3− and placebo. Placebo had no effect but NO3− led to an 8.6-fold increase in plasma NO2−, which was accompanied by an increase in FMD , and a reduction in AIx75 . PLM hyperaemia, BP, and PWV were unchanged during both trials. This study showed that a dose of dietary NO3− improved NO bioavailability and enhanced endothelial function as measured by femoral artery FMD. These findings provide insight into the specific central and peripheral vascular responses to dietary NO3− supplementation in older adults. Aging is often associated with reduced leg blood flow, increased arterial stiffness, and endothelial dysfunction, all of which are related to declining nitric oxide (NO) bioavailability. Flow mediated dilatation (FMD) and passive leg movement (PLM) hyperaemia are two techniques used to measure NO-dependent vascular function. We hypothesised that acute dietary nitrate (NO Cardiovascular diseases are the leading cause of morbidity and mortality worldwide  and ther3−), such as beetroot (Beta vulgaris), can be reduced to nitrite (NO2−) by oral bacteria  and [NO2−] was carried out by gas-phase chemiluminescence using a nitric oxide analyser and following manufacturer’s instructions  [2−], potassium iodide in acetic acid was used as the reductant, which converts NO2− to NO but does not reduce higher oxides of nitrogen such as NO3−. Prior to the measurement of plasma [NO3−], samples were deproteinated using zinc sulphate (ZnSO4)/sodium hydroxide (NaOH) precipitation. This was performed by adding 200 µL of plasma to 400 µL of ZnSO4 (10% w/v) and 400 µL of 0.5M NaOH. The solution was vortexed for 30 s and left to stand at 22°C for 15 min, centrifuged at 4000 g for 5 min and the supernatant fraction was collected and used for determination of [NO3−]. The [NO3−] was determined using a reductant of vanadium chloride in 1 M HCl at 90°C. The [NO3−] and [NO2−] in samples was calculated by converting the nitric oxide analyser signal into analyte concentration using a standard curve of known concentrations of sodium NO3− or NO2−. The area under the curve was determined using Origin software (version 7.1).Plasma samples were thawed at room temperature (21 °C) in the dark and all assays were performed within 10 min of thawing. Measurement of plasma [NOCO, USA) . To dete3− on superficial femoral artery FMD and passive leg movement hyperaemia has not been previously described, sample size was calculated from data presented by Rammos et al [3−, brachial FMD increased from 6.0 ± 0.8% to 6.5 ± 0.8%, for older adult males (63 ± 5 years). Assuming a similar effect size (0.79) and variance, with power at 0.99 and an alpha level of 0.05, we determined a priori that 10 participants in a cross-over design would be needed to demonstrate a significant effect [As the effect of dietary NOos et al . Followit effect . To allo3− vs. placebo days for all variables. Where ANOVA revealed a significant F test, pair-wise comparisons were examined to identify the specific location of differences. Where delta data were used to compare NO3− vs. placebo trials, one-way ANOVA was used. In addition, to conform with recent recommendations for FMD analysis [p < 0.05. All data are expressed as mean ± standard deviation or 95% confidence intervals. Investigators were blinded to test condition until all image and blood flow analyses were complete. Statistical analyses were performed using SPSS . Two-factor (time × condition) mixed ANOVA for repeated measures was used to compare the NOanalysis , statistanalysis , therefoanalysis . StatistParticipant characteristics are displayed in 3− or plasma NO2− with placebo; however, following NO3− supplementation, there was a 16.1-fold  increase in circulating plasma NO3− and an 8.6-fold  increase in plasma NO2− compared to pre-NO3− , providing a consistent baseline measure for FMD tests. Reactive hyperaemia peak flow rate and AUC were similar on all occasions, as was the response of shear rate. Flow mediated dilatation was not different pre- to post-placebo; however, FMD was significantly increased post-NO3− compared to pre-NO3− (AUC−1), and when it was expressed as percent change  compared to post-NO3− (181 ± 116 mL·min−1) and the placebo trial (pre: 253 ± 189 mL·min−1 and post: 163 ± 148 mL·min−1), p = 0.048. During passive leg movement, there was no difference in the hyperaemic response pre or post in the NOesponses . Mean di3− supplementation would improve vascular function in older adults. The main findings are that an acute dose of dietary NO3− increased plasma NO2−, suggesting increased NO bioavailability, and this was associated with enhanced lower limb FMD and improved (lower) pressure augmentation index. However, dietary NO3− did not alter pulse wave velocity or the hyperaemic response to passive leg movement. The primary aim of this placebo-controlled, double-blind, cross-over design study was to determine whether NO3− supplementation on vascular function in the leg began with the observation that NO3− supplementation was associated with a decrease in blood pressure [3− may enhance plasma NO2−, which may increase NO bioavailability [Our interest in the impact of NOpressure  and an ipressure , and in pressure ,24, hearpressure , and stapressure . It was lability . Nitric lability  and may lability  by incre3− supplementation with an increase in blood plasma NO3− and NO2− while there was no change in plasma NO3− or NO2− during the placebo trial. The link between NO3− ingestion, circulating plasma NO2−, and NO bioavailability has been clearly defined [3−, dietary restriction prior to or during the experiment, oral microbiome and oral hygiene, medication and supplement use, and fitness status [2− following NO3− supplementation. This increase is similar in magnitude to other well-controlled studies investigating NO3− supplementation in older adults [All participants responded to NO defined , but inds status ,64,65. Wr adults ,66 and i3− supplementation. Four previous studies with varied NO3− doses (180–397 mg·day−1 and 9.3 mg·kg body weight−1), sources , and treatment schedules (acute vs. ongoing) have indicated that NO3− supplementation can increase brachial artery FMD in older adults [3− improves FMD in the superficial femoral artery, reflecting an enhanced endothelial function in the legs of older adults. Our results demonstrate that FMD in the superficial femoral artery increased following NOr adults ,29,30,35r adults ,12,37, ar adults , the thir adults , or it mr adults ,12,36. F3− supplementation on brachial FMD in older adults report inconsistent findings, with some indicating an increase as noted above [3− on FMD included participants who did not take medication and were free from disease [3− supplementation included participants who were taking anti-hypertensive and anti-platelet medications. Anti-hypertensive medications are likely to have a vasodilating effect [3− supplementation. Another possible explanation for the null findings is that these two studies included participants who had been diagnosed with peripheral arterial disease [3− supplementation for individuals with cardiovascular disease. Previous studies of the effect of NOed above ,29,30,35ed above ,68. Simi disease ,30,35,69g effect  which ma disease  and type disease ,34, whic disease , however disease ,71. Rega3− supplementation, suggesting there was no change in vasodilation of arterioles downstream from the site of cuff occlusion. Importantly, this indicates that the increase in FMD was not a result of increased shear stress. One explanation for this finding is the dampening effect that dietary NO3− may have on the abundance of reactive oxygen species [3− supplementation enhanced the antioxidant defence system [3−–NO2−–NO pathway. This theory is supported by evidence of improved FMD following antioxidant treatments, such as vitamin E [3− from vegetables such as beetroot may reduce oxidative stress more than individual vitamins or minerals because it may simultaneously restore redox imbalance and enhance NO generation [Reactive hyperaemia did not increase following NO species ,73. Elev species ,75. It ie system ,77, redue system , rather itamin E ,79 and vitamin E . Dietaryneration . This pr3− supplementation. Passive leg movement hyperaemia is measured at the common femoral artery, which typically does not dilate during this assessment, and the hyperaemic response is understood to reflect downstream arteriole endothelium-dependent flow regulation [3− supplementation suggests that passive leg movement hyperaemia may not be a sensitive test for fluctuations in NO bioavailability in older adults. Collectively, our findings of increased FMD, unchanged reactive hyperaemia, and unchanged passive leg movement hyperaemia, indicate that, in the leg, conduit artery function is sensitive to a dose of dietary NO3−, while downstream arteriole flow regulation is not. While the increase in FMD following NO3− supplementation indicates an improvement in lower limb endothelial function, the lack of change in reactive hyperaemia and passive leg movement hyperaemia raises questions about the functional relevance of this finding. The impact of dietary NO3− on femoral blood flow during exercise has not been described in older adults. Given that conditions of low oxygen tension and low pH that may be observed during exercise promote reduction of NO2− to NO, future studies should investigate whether exercise blood flow responses are augmented with dietary NO3−. Passive leg movement hyperaemia, a recently established measure of NO-dependent vascular function ,42, did gulation . Passivegulation ,81, but 3− supplementation on passive leg movement hyperaemia is insufficient stimulation. Studies exploring NO3− supplementation in hypoxia demonstrate that the conversion of NO2− to NO is facilitated by perturbations to homeostasis, such as a reduction in oxygen tension [2− did not increase the hyperaemic response to passive leg movement because metabolic stress is necessary to activate mechanisms that produce NO from NO2−.Another explanation for the absence of an effect of NO tension ,82,83. I tension ,41,84,85 tension , reveali3− and pre- and post-placebo, however resting flow was lower pre-NO3−. Trial order was randomised for each participant and this measure was taken prior to ingestion of NO3−. There was no reason that we can identify to explain why leg blood flow was lower pre-NO3−. Resting leg blood flow, assessed immediately prior to the onset of passive leg movement, was similar post-NO3− and placebo trials, although the difference only reached significance following administration of dietary NO3−. The time-related effect may reflect a redistribution of blood volume and reduced cardiac preload [3− suggests a temporary cardiovascular benefit. Endothelial function and arterial stiffness are related [3− supplementation in older adults [Augmentation index tended to be lower in the afternoon during both the NO preload  or there preload ,89, as a preload . For thi preload ,92 and a related  and meas related . The facr adults ,29,30.3−. This concurs with some [3− supplementation. There are equivocal reports in the literature regarding whether dietary NO3− reduces blood pressure in older adults [Pulse wave velocity was unchanged by a dose of dietary NOith some ,95,96 buith some ,29,30. Sr adults ,97. Therr adults . However3− supplementation. Some participants in our study were taking low dose statins and, in addition to lowering cholesterol, statins may improve endothelial NOS expression and improve baseline NO bioavailability [2− was low, even among those who were taking statins, suggests low NO status irrespective of medication use. During the study, there were no changes to the use of statins, or schedule or timing of dose for any individual, and experiment trials were carried out on the same day and time, separated by one week. Further, the cross-over design enabled participants to act as their own controls. Participant use of medication for cardiovascular conditions may mask the effect of NOlability . The fac3− supplementation [2− in females, which is associated with lower blood pressure [2− in females leads to a saturation effect that may reduce sensitivity to NO3− supplementation compared to men [Sex differences have been noted in the literature regarding the effectiveness of dietary NOentation ,102,103.pressure . This ispressure  and it ipressure . It has d to men . This in3− increased plasma NO2− and FMD in the conduit vessels of the leg. Future research should investigate whether there is an association between improvements in leg FMD and improvements in exercise tolerance for older adults following NO3− supplementation. In this study of older adult males, dietary NO


We have succeeded in preparing various water-soluble metal phthalocyanine (MPc)–polymer complexes, wherein the metal moiety is lithium, iron, cobalt, copper, zinc, or tin, and the polymer is one of the following water-soluble polymers: polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), or polyvinyl alcohol (PVA). Among all MPc–polymer complexes, the iron phthalocyanine (FePc)–PVP complex in water showed the largest and sharpest absorption peak at ∼700 nm in UV–Vis absorption spectrum, which indicates that FePc–polymer complexes in water are easily prepared and the degree of stacking of FePc in the complexes, very small, such as that of a monomer or a similar structure. Conversely, the polymer chains including those of PEG, PVP, and dextran have high biological affinity as well as flexibility. Speculatively, the FePc–polymer  complexes adsorbed onto the surface of a cancer cell might break it via the irradiation of near-infrared light having a wavelength of ∼700 nm. Furthermore, chlorophyll a–polymer complexes, previously prepared by our group, might similarly break a cancer cell because these complexes showed a large and sharp absorption peak at ∼700 nm in UV–Vis spectrum. However, since MPcs are insoluble in these organic solvents, water-soluble MPc–polymer complexes cannot be prepared using this route and these organic solvents.As known well, metal phthalocyanines (MPcs) and chlorophyll a are hydrophobic substances and therefore insoluble in water. In previous studies, we were successful in preparing the water-soluble chlorophyll a–polymer complexes by using the water-soluble polymers such as polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), dextran, or polyvinyl alcohol (PVA) , 4, 5, 6b, such as pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or N,N-dimethylacetamide (DMA). Using these organic solvents, we successfully prepared the various water-soluble MPc–polymer complexes. The water-soluble MPc–polymer complexes obtained probably have the potential of being used in numerous fields.Alternatively, it is well known that MPcs dissolve in organic solvents with a very high value of Tet al. [Recently, H. Kobayashi et al. , 8, 9 suSpeculatively, FePc–polymer  complexes adsorbed onto the surface of a cancer cell might similarly break the cancer cell via irradiation process of the near-infrared light having a wavelength of ∼700 nm because, in the present study, the FePc–PVP complex in water exhibited the largest and sharpest absorption peak at ∼700 nm in the UV–Vis absorption spectrum among all the MPc–polymer complexes. Herein, it is considered that the largest and sharpest absorption peak indicates that FePc–polymer complex can be easily formed, and thus can be easily prepared, and the degree of stacking of FePc in the complexes is very small, such as that of a monomer or a similar structure. Similarly, chlorophyll a–polymer  complexes might break cancer cells via irradiation of near-infrared light because the complexes in water showed a large and sharp absorption peak at ∼700 nm , 4, 5, 622.12Pc, FePc, CoPc, CuPc (β-form), ZnPc, and SnPc. These MPc samples were purchased from Tokyo Chemical Industry Co., Ltd. and were used without further purification. Since MPcs have a conjugated double bond system, these species tend to stack with each other by way of π–π interaction.As shown in PEG  and PVP  were purchased from Kishida Chemical Co., Ltd. PVA  was donated from Kuraray Co., Ltd. Notably, PVA can be dissolved in water at a temperature higher than 95 °C.b = 116 °C), DMSO (Tb = 189 °C), DMF (Tb = 153 °C), and DMA (Tb = 166 °C (754 mmHg)) were purchased in JIS special grade (purity ≥99%) from Kanto Chemical Co., Inc. and used without further purification.Pyridine 2Pc = 526 (minimum)molecular weight: Li2Pc (0.01 g) = 0.01/526 mol = 1.9 × 10−5 mol∴ Limolecular weight: Sn(II)Pc = 631 (maximum)−5 mol∴ Sn(II)Pc (0.01 g) = 0.01/631 mol = 1.6 × 10−5 molmolecular weight: PEG = 20,000 ∴ PEG (1 g) = 1/20,000 mol = 5 × 102Pc = 5 × 10−5/1.9 × 10−5 = 2.6molar ratio: PEG/Li−5/1.6 × 10−5 = 3.1molar ratio: PEG/Sn(II)Pc = 5 × 10∴ molar ratio: PEG/MPc = 2.6–3.1 ∴ molar ratio: PEG/MPc = 3.0MPc (0.01 g), polymer  (1 g), and organic solvent  (5 g) were mixed in a beaker, and the resulting mixture was gently stirred with a micro-spatula at 40 °C (for PEG and PVP) or at 95 °C (for PVA), until a homogeneous paste was formed. At this stage, the molar ratio of polymer/MPc is 3.0 in the case of PEG, 1.5 in the case of PVP, and 2.4 in the case of PVA. As an example, we explain the method for calculating the molar ratio of PEG/MPc as follows:Step (2)b of the organic solvent).2 g of the paste was put into a test tube. The test tube was then placed in a silicone oil bath kept at a constant temperature (10–20 °C below the TStep (3)The paste was then dried under a reduced pressure (1.7 kPa) using an aspirator. At this time, the paste was quickly transformed to a porous solid material consisting of MPc and polymer.Step (4)The temperature of the silicone oil bath was reduced to 40 °C (in the cases of PEG and PVP) and 95 °C (in the case of PVA). Subsequently, air was allowed to flow into the test tube until the pressure on the test tube reached the atmospheric pressure.Step (5)A small amount of water was then added to the solid present in the test tube, and the resulting mixture was gently stirred with a micro-spatula at 40 °C (in the case of the mixture containing PEG or PVP) and 95 °C (in the case of the mixture containing PVA) until a homogeneous paste consisting of MPc, polymer, and water was obtained.Step (6)The paste in the test tube was diluted with water to a given concentration and the test tube was removed from the silicone oil bath. As a result, a mixture comprising a water-soluble MPc–polymer complex was obtained at room temperature.Step (7)The mixture thus obtained was filtered through a nitrocellulose-type membrane filter having a pore size of 0.45 μm. At this point, an aqueous solution of the MPc–polymer complex was obtained successfully. The color of this solution was blue for all the complexes that were synthesized.Note (1)b of the solvents utilized in this study. Therefore, the aqueous solution of the MPc–polymer complex obtained toward the end of Step (7) may still contain small amounts of the organic solvent. In fact, when pyridine was used as the organic solvent, the filtered solution obtained in Step (7) smelled like pyridine. However, using a strong vacuum pump at Step (3), the organic solvent molecules will be removed almost completely. Furthermore, it is strongly expected that almost all the organic solvent molecules will be removed by the dialysis membrane method after Step 7.The porous solid material obtained in Step (3) may still contain a small amount of organic solvent molecules owing to a very high value of TNote (2)b for pyridine is the lowest among used organic solvents and the presence of even a small amount of pyridine in the MPc–polymer complex aqueous solution can be easily detected by smell.Pyridine is strongly recommended as an organic solvent to dissolve MPc because the value of T2.3UV–Vis absorption spectra of the MPc–polymer complexes in water and those of MPcs in organic solvents were measured using a double beam spectrophotometer, UVIDEC-510 , at room temperature.33.12Pc, FePc, CoPc, CuPc, ZnPc, or SnPc, and the polymers are PEG, PVP, or PVA. The figure also shows the spectra of various MPcs dissolved in organic solvents . In particular, the thin curves are the spectra of MPcs in organic solvents, whereas the thick, bold curves are the spectra of MPc–polymer complexes in water. For clarity, the two spectra of each MPc series are shifted upwards along the vertical axis direction by a quantity of A, where A = 0, 1, 2, 3, 4, or 5.2Pc, FePc, CoPc, CuPc, ZnPc, or SnPc)–polymer  complex was prepared.The UV–Vis spectra of all MPc–polymer complexes in water are characterized by two or three absorption peaks in the 500–800 nm wavelength range. This indicates that each water-soluble MPc  an increase in the degree of stacking of MPc in the complexes as compared with that of the MPc in organic solvent, (2) interactions between MPc and polymer chain, and (3) a change in the solvent of MPcs or MPc–polymer complexes from the organic solvent to water, i.e., solvatochromism, which is the phenomenon observed when the color caused by a solute changes when that particular solute is dissolved in different solvents.The peaks due to the complexes LiNotably, in Generally, the absorption peak at ∼700 nm due to MPc or chlorophyll a in the complexes is assumed to become sharper with the decrease in the degree of stacking of MPc or chlorophyll a , 4, 5, 6Additionally, we experimentally confirmed that the mentioned absorption peak at ∼700 nm, which is due to the MPc–polymer complex in water, only underwent small changes when the polymer used to prepare it changed.3.22–CH2– or (–CH2–CH–)n) and the hydrophobic substance of MPc. On the contrary, the hydrophilic part of the polymer (ether oxygen atoms or pyrrolidone groups) interacts via hydrogen bonds with the water molecules present around the complexes. Consequently, the MPc–polymer complexes dissolve in water. In the case of the water-soluble chlorophyll a–polymer complexes, a similar mechanism can be suggested.The MPc–polymer (PEG or PVP) complexes form as a consequence of the hydrophobic interaction between the hydrophobic part of the polymer . As mentioned in the section of 1. Introduction, the chlorophyll a–polymer complexes can be very easily prepared at room temperature [Although our group isolated chlorophyll a from fresh spinach leaves , 4, 5, 6perature , 4. Addiin vivo; thus, it may remain on the surface of cancer cell for a sufficiently long period to allow it to break the cancer cell via irradiation of near-infrared light.The rationale for considering these complexes as a potential tool to break cancer cells is as follows: (1) the degree of stacking of FePc or chlorophyll a in the complex is very small (corresponding to that of a monomer or a similar structure); (2) because FePc or chlorophyll a is wrapped by polymer chains with high biological affinity , 11, 12 Recently, polymeric nanostructured materials, e.g., micelles, nanogels, polymersomes, nanoparticles, nanocapsules, nanofibers, dendrimers, and nanocomposites have been studied in detail for the medical applications , 14, 15.However, no experiments involving cancer cells and cancer cell lines have been performed at present. Inspections by these experiments are required to be conducted toward the application for cancer therapy. Furthermore, for validation, all complexes must be characterized with further analytical techniques  along with their UV–Vis absorption spectra.While experimenting with cancer cells and cancer cell lines, if none of the aforementioned complexes can break these cells, more suitable polymers, which contain newly synthesized ones, must be searched to break them , 14, 15.Isamu Inamura: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Kentaro Inamura, Yuji Jinbo: Analyzed and interpreted the data; Wrote the paper.Toshinao Mihara, Yasuhiro Sasaoka: Performed the experiments.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


Streptomyces coelicolor strain A3(2) strain to convert valencene to the grapefruit metabolite nootkatone, and confirmed its enzymatic activity by gas chromatography-mass spectrometry. This enzyme re-positioning approach may offer an entirely new way of screening enzymes that cannot be achieved by most of other conventional methods, and it is applicable to various other metabolites and may enable microbial production of compounds that are currently difficult to produce industrially.Plant-biosynthesised secondary metabolites are unique sources of pharmaceuticals, food additives, and flavourings, among other industrial uses. However, industrial production of these metabolites is difficult because of their structural complexity, dangerousness and unfriendliness to natural environment, so the development of new methods to synthesise them is required. In this study, we developed a novel approach to identifying alternative bacterial enzyme to produce plant-biosynthesised secondary metabolites. Based on the similarity of enzymatic reactions, we searched for candidate bacterial genes encoding enzymes that could potentially replace the enzymes in plant-specific secondary metabolism reactions that are contained in the KEGG database (enzyme re-positioning). As a result, we discovered candidate bacterial alternative enzyme genes for 447 plant-specific secondary metabolic reaction. To validate our approach, we focused on the ability of an enzyme from   •We developed ‘bacterial enzyme gene re-positioning’ that is a method to search for enzyme genes based on reaction similarity.•We identified candidate bacterial alternative enzymes for 447 plant-specific secondary metabolism reactions.•An enzyme for synthesising nootkatone from valencene was discovered. For exaTo overcome the challenges of extracting metabolites from plants, several means of synthesising metabolites artificially have been developed. Chemical synthesis is popular methods of stably obtaining metabolites that do not involve extraction from plants. Nonetheless, organic synthesis is not always readily possible, because plant secondary metabolites have complex structures and large molecular sizes. In such cases, organic synthesis requires many steps to obtain target metabolites from simple substrates, constituting a great hurdle for industrial large-scale synthesis of plant-specific secondary metabolites . On the 2), which has an environmental impact  (Mucor (fungi) (Rhodococcus (bacteria)  . Further   and meta (fungi)  and Rhodacteria)  have beeEscherichia coli, identified CYP109B1 from Bacillus subtilis that produces nootkatone from valencene  and producing plant-specific secondary metabolites, yet this approach is extremely difficult because of decreasing expression level and enzyme activity due to differences in biological function . In receIn this study, we developed a method to search for genes that encode enzymes based on similarity of enzyme reactions and not on gene sequences. In other words, we aimed to search for enzymes with similar metabolic reactions but with dissimilar gene sequences. Furthermore, in order to validate the approach, we discovered an enzyme for synthesising nootkatone from valencene that has no sequence similarity with the known plant enzyme.22.1We used KEGG OC  database2.2Streptomyces lividans TK64 (NBRC 15678), which was purchased from National Institute of Technology and Evaluation , was used as the host organism for bioconversion. S. lividans TK64 was cultured in TSB medium ; BD, Heidelberg, Germany) supplemented with 50 μg/mL of thiostrepton at 28 °C and 200 rpm for 72 h.Escherichia coli JM109  was used as the host organism for constructing plasmid clones. Escherichia coli JM109 was cultured in LB medium  at 37 °C for 16 h.2.3Streptomyces coelicolor A3(2) (GenBank No. NC_003888), SCO5223, SCO3099, SCO3770 and SCO3636 were cloned. The genomic DNA of S. coelicolor A3(2) was acquired by phenol-chloroform extraction. The coding sequences of four genes were amplified with primers (S. coelicolor A3(2) genomic DNA as a template. PCR was performed under the following conditions: 94 °C for 2 min, followed by 30 cycles of 98 °C for 10 s and 68 °C for 1.5 min . The PCR products were purified and digested with SphI, except for the SCO3636 PCR product, which was digested with EcoT22I. pIJ702D, a derivative of pIJ702SSMP from which melC1 and melC2 have been removed (SCO3636)  . The PCRSCO3770 partially-knockout vector, nucleotides 97 to 1090 of SCO3770, which encode an inactive form of the enzyme, were amplified with primers (S. coelicolor A3(2) genome as a template. PCR was performed under the following conditions: 98 °C for 30 s, followed by 30 cycles of 98 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min . The PCR product was purified and digested with EcoRI and KpnI. pk18melC, a derivative of pk18mob carrying melC1 and melC2 was digested with the same restriction enzyme, and the fragments were ligated together  supplemented with 10 μg/mL of thiostrepton and incubated at 28 °C and 160 rpm. After 72 h, glucose and valencene  were added at final concentrations of 10% and 0.025%, respectively. Cultivation was then continued for another 168 h.2.6g for 20 min. The cell pellet was resuspended in 1.5 mL ethanol, and 0.25 mL distilled water and 0.5 mL n-hexane were added to 1.0 mL of the supernatant and mixed by shaking. The n-hexane layer was collected by centrifugation at 14,000×g for 1 min. Nootkatone in the extract was analysed by GC/MS . The peak of nootkatone was at approximately 12.0 min. Measurements were performed under the conditions indicated in Following culture of the P450-overexpressing strains, cells were harvested from 2 mL whole broth by centrifugation at 14,000×2.7S. lividans TK64, S. lividans TK64/pIJ702D:SCO3770, S. lividans TK64 ΔSCO3770, and S. lividans TK64 ΔSCO3770/pIJ702D:SCO3770) were cultured, and cells were harvested from 2 mL whole broth by centrifugation at 14,000×g for 20 min. Next, 0.5 mL n-hexane was added to 1.0 mL of the supernatant and mixed by shaking, after which the n-hexane layer was separated by centrifugation at 14,000×g for 1 min. The cell pellet was resuspended in 1.5 mL ethanol, and 0.25 mL distilled water and 0.5 mL n-hexane were added to 1.0 mL of the supernatant and mixed by shaking. Nootkatone in both extracts was analysed by GC/MS  under the conditions indicated in Four strains , which is a representative Streptomyces species and generally used for enzyme screening, exhibited 365 reactions with a similarity score ≥ 0.4, and 43 reactions with a similarity score ≥ 0.9 , which belong to the CYP170A, CYP107U, CYP107P and CYP107T subclasses of the P450 family, respectively, as alternative enzyme candidates for nootkatone biosynthesis according to their similarity scores (E1B2Z9 (Cichorium intybus) and B8ZIU7 (Pleurotus sapidus), the total scores (BLAST bit score) compared to four genes were low, less than 54.6 and identity was also low, less than 30%. For the prokaryotic-derived gene U5U1Z3 (Bacillus subtilis), the BLAST bit score was 99–229, and some homology with four genes was observed, even though identity was only 24–36%. Thus, it could never be considered that these scores were top hits for sequence similarity based screening.We detected four y scores . Our enzy scores  . First, SCO3770 overexpressing (S. lividans TK64/pIJ702D:SCO3770), genomic SCO3770 homolog partially-knockout (S. lividans TK64 ΔSCO3770) and SCO3770 complementation (S. lividans TK64 ΔSCO3770/pIJ702D:SCO3770) strains were constructed. Next, these three strains and wild-type S. lividans were cultured in medium containing valencene for 168 h, the culture supernatant was extracted with hexane solvent, and the nootkatone in hexane was detected by GC-MS . These results indicate that SCO3770 is involved in the conversion of valencene to nootkatone.To confirm the involvement of by GC-MS . Althoug4In this study, we developed ‘bacterial enzyme gene re-positioning’ that is a method to search for enzyme coding genes based on similarity of enzyme reactions, and identified candidate bacterial alternative enzymes for 447 plant-specific secondary metabolism reactions. In addition, an enzyme for synthesising nootkatone from valencene, SCO3770 was discovered by our approach, and SCO3770 had low sequence homology with existing enzymes. This result showed that our ‘bacterial enzyme gene re-positioning’ approach has a novel enzyme screening space which is not considered by sequence based similarity approach, and it may enable us to produce compounds that are currently difficult to produce industrially by microbial fermentation.Our bacterial enzyme gene re-positioning approach detected candidate bacterial alternative enzymes for 447 plant-specific secondary metabolism reactions, and 41 of 447 (9.2%) of the plant-specific secondary metabolism reactions for which re-positioning candidates were identified involved “orphan enzymes”, whose reactions are reported but the genes are not discovered . AlthougOf the plant-specific orphan enzymatic reactions for which we found alternative enzymes in this study, about half of the 19 reactions belonged to flavone-related metabolic pathways. Flavone-related compounds reportedly have anti-tumour  and antiOur result of phylogenetic distribution analysis for discovered candidate bacterial alternative enzymes indicated that the bacterial enzyme candidate genes clustered into a few taxa. The bacterial enzyme candidates were phylogenetically biased, with Pseudonocardiaceae, Streptomycetaceae, Mycobacteriaceae, Myxococcales and Burkholderiaceae possessing more alternative enzyme candidates than other taxa. Since Streptomycetaceae and Burkholderiaceae have particularly many secondary metabolic functions among all of bacteria, it is possible that such bacterial secondary metabolism related enzymes have been detected as alternative candidates for plant secondary metabolism. Moreover, all these bacteria live in soil , and sinIn this study, we applied this method to plant secondary metabolism, however the scope of application of this method is not limited to plant reactions. It is possible to search for the enzyme reactions of all organisms. Not only that, it is possible to search for reactions in which no enzyme has been found if we have only a substrate-product pair. Therefore, applying this method may enable us to flexibly search for the enzyme genes for the desired reaction. This method may be used to search for enzymes for producing industrially valuable compounds from inexpensive compounds without considering whether the reactions are catalysed by the enzymes. the reactions.5Taken together, we developed a novel method, bacterial enzyme gene re-positioning, to efficiently screen bacterial alternative enzymes for plant-specific secondary metabolism based on reaction similarity and found a nootkatone biosynthetic enzyme and many alternative enzyme candidates for other reactions. This bacterial enzyme gene re-positioning method may offer an entirely new way of screening enzymes that cannot be achieved by most of other conventional methods. Overall, this method will enable the production of compounds that are difficult to synthesise. In the future, the discovery of new alternative enzymes through application of this methodology will enable microbial fermentation-mediated mass production of various compounds.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Y. M., S. H. and T. Y. designed this study. Y. N. and Y. M. performed the bioinformatic screening and other bioinformatic analysis. Y. T. performed the bacterial culture and other experiments. T. Y. and S. H. provided senior guidance. Y. N. wrote the manuscript with assistance from other authors. All authors reviewed and approved the final version of the paper.No conflict of interests is declared.


P = 0.2), mortality rate  and recurrent MI occurrence , with no evidence of heterogeneity, when given to patients with AMI. Cyclosporine also did not significantly lessen the rate of rehospitalisation in AMI patients when compared to placebo , with moderate heterogeneity (I2 = 46%). There was also no significant improvement in heart failure events between Cyclosporine and placebo in AMI patients . No serious adverse events were reported in Cyclosporine group across all studies suggesting that Cyclosporine is well tolerated when given to patients with AMI. The use of Cyclosporine in this group of patients, however, did not result in better clinical outcomes vs. placebo at improving LVEF, mortality rate, recurrent MI, rehospitalisation and heart failure event.There are various studies that have addressed the use of Cyclosporine among patients with acute myocardial infarction (AMI). However, to date there is hardly any concise and systematically structured evidence that debate on the efficacy and safety of Cyclosporine in AMI patients. The aim of this review is to systematically summarize the overall evidence from published trials, and to conduct a meta-analysis in order to determine the efficacy and safety of Cyclosporine vs. placebo or control among patients with AMI. All randomized control trial (RCT) published in English language from January 2000 to August 2017 were included for the systematic review and meta-analysis. A total of six RCTs met the inclusion and were hence included in the systematic review and meta-analysis. Based on the performed meta-analysis, no significant difference was found between Cyclosporine and placebo in terms of left ventricular ejection fraction (LVEF) improvement (mean difference 1.88; 95% CI −0.99 to 4.74;  Cardiovascular disease poses tremendous burden on public health, as well as the global economy. According to the World Health Organization, an estimated 17.5 million people died from cardiovascular disease in 2012, accounting for 31% mortality globally WHO,  with an Reperfusion therapy, either by percutaneous coronary intervention (PCI) or intravenous fibrinolysis therapy, remains the mainstay therapeutic management of STEMI  was performed from January 2000 to August 2017. Studies selected for our systematic review and meta-analysis were based on criteria as follow:All articles published in English that were published in PubMed, Embase, and CINAHL databases from January 2000 to August 2017 were searched using the search terms; The following words were combined with “cyclosporin”, using the Boolean operators (“AND” & “OR”): “AMI”, “MI”, “cardiovascular stroke”, “myocardial infarct”, “PCI”, “Percutaneous coronary intervention”, “infarct size”, “ischaemia”, “myocardial stunning”, “ciclosporin”, “Cyclosporine-Neoral”, “Cya-NOF”, “Cyclosporine A”, “Neoral”, “OL-27-400”, “Sandimmun”, “Sandimmun Neoral”, “effectiveness”,“treatment outcomes”, “reperfusion injury”, “clinical effectiveness”, “clinical efficacy”, “rehabilitation outcome”, “treatment effectiveness”, and “treatment efficacy”.We included all RCT with a control group receiving either placebo, or no intervention, and were published within the timeline and databases mentioned above. Other study designs i.e., non-RCTs, reviews, letters, case studies, conference papers, animal studies, reports or editorial, non-English literatures, report not found in print or online domain, or qualitative studies, were excluded.Potential studies were identified by using following keywords or subject heading: (1)AMI: “AMI” OR “MI” OR “cardiovascular stroke” OR “myocardial infact” OR “myocardial stunning”; (2) Cyclosporine: “Cyclosporine” OR “Ciclosporin” OR “Cyclosporine-Neoral” OR “Cya-NOF” OR “Cyclosporine A” OR “Cyclosporine A” OR “Neoral” OR “OL-27-400” OR “Sandimmun” OR “Sandimmun Neoral” OR “Sandimmune”; (3) Efficacy: “effectiveness” OR “treatment outcomes” OR “clinical effectiveness” OR “clinical efficacy” OR “patient-relavant outcome” OR “rehabilitation outcome” OR “treatment effectiveness” OR “treatment efficacy”; (4) Safety: “Safety” OR “Patient Harm” OR “Patient safety” OR “ADR” OR “adverse drug” OR “adverse drug reaction” OR “side effect.”Two investigators then independently screened the identified studies; Data extraction and risk assessment of the studies were also done by all investigators independently. The Cochrane Risk of Bias Assessment tool for RCT was used to assess the quality of all included studies  presenting with AMI. Studies that involved the participants without AMI, and children were excluded from this overview.We selected all studies that used Cyclosporine as their intervention regardless of dosages, formulations, and administration routes used.The data was searched for outcomes in the following categories; (i) the efficacy of Cyclosporine administered in patients with AMI measured by: the left ventricular ejection fraction (LVEF), mortality and rehospitalisation secondary to worsening heart failure, recurrent MI, heart failure occurrence and infarct size  and cardiac troponin I or T (cTnI or cTnT) or MRI/ CMR); (ii) the safety of Cyclosporine used in patients with AMI.2 value of more than 75% is a considerable heterogeneity, while I2 value below than 40% suggest that heterogeneity might not be important between studies  between clinical studies for a comparison of LVEF percentage after administration of Cyclosporine in patients with AMI. Mean difference values smaller than 0 indicate a result that favors Cyclosporine compared to placebo. Odds ratios (ORs) were calculated for other outcomes such as mortality rate, rehospitalization, MI recurrence, and heart failure occurrence for comparison between clinical studies. Odds ratio values < 1 favor Cyclosporine over placebo. Fixed-effect model was used to calculate pooled MD and ORs. For each pooled MD and OR, we performed the equivalent z test and the results were considered statistical significant if p value < 0.05. Heterogeneity Ies Ryan, . PublicaWe identified 2,037 articles, of whom 293 were duplicates. Based on inclusion and exclusion criteria, 1686 were excluded. Finally a total of 58 papers were subjected to final screening. Further screening based on title and abstract, excluded 31 papers, resulting in retrieval of 27 full papers. Of the 27 studies, 10 were excluded for not meeting the population inclusion criteria, and a further 1 1papers were excluded because they were not RCTs . Finally, only 6 randomized trials met all the inclusion criteria and were hence included in this systematic review. Data obtained during literature search process was presented using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flowchart  in their treatment arms. The duration of studies varied from hospitalization stay to 1 year. The studies had different length of outcome follow up. The shortest follow up were reported in study by Piot et al. ,48 hoursThe summary risk of bias for included studies is shown in Figures The description of outcomes for each study is illustrated in Table p = 0.53), length of stay in the postoperative critical care unit (p = 0.53) and inotrope use. The balloon pump also was not needed for inotropic support in any of the patients postoperatively. Two patients (6%) in the Cyclosporine group and none of the patients in the control group presented with a postoperative wound infection.Postoperative outcome measures showed that there were no significant differences in the length of hospital stay (p = 0.04) corresponding to 26 and 36% reductions in AUCs for creatinine kinase and troponin I release, respectively.Measurement of the area of delayed hyperenhancement , using cardiac magnetic resonance imaging (MRI) in a subgroup of patients, which was assessed on day 5 after infarction, showed that Cyclosporine significantly reduced the absolute mass of the area of hyperenhancement for about 20% as compared with the control group (P = 0.05).For other end points observed during the first 48 hours after reperfusion, seven adverse clinical events were recorded in the control group (one episode of ventricular fibrillation and six episodes of heart failure) as compared with three adverse clinical events in the Cyclosporine group . When only infarct related events were considered , seven events were observed in the control group vs. two in the Cyclosporine group  between the 2 groups. Results on LV regional wall thickness showed no significant difference between the 2 groups in either the global LV mass or regional wall thickness of the remote non-infarcted myocardium or infarcted myocardium of patients, at 5 days and 6 months.P = 0.80], occurrence of heart failure , LVEF at admission  and discharge , and TLT-related complications . In-hospital  and 6-month  mortality rates were similar between groups.In the study by Ghaffari et al. , the endP = 0.69], heart failure , or mean LVEF at admission , and mean LVEF at discharge  between treatment vs. control groups. Two patients in each group died at hospital and during the follow-up period (P = 1), among this sub-group of patients. Therefore, the pre-thrombolytic administration of Cyclosporine was not associated with a reduction in the infarct size or any improvements in clinical outcomes.Repeated analysis was done in subgroups of patients and again, there was no significant difference in occurrence of arrhythmic events .Cyclosporine is an immunosuppressive agent that is often associated with adverse events after chronic use, such as nephrotoxicity, hypertension, hyperlipidaemia, neurotoxicity, hepatotoxicity, anorexia, nausea, vomiting, paraesthesia, hypertrichosis, gingival hyperplasia, and tremor (Hausenloy et al., The cumulative evidence from our systematic review and meta-analysis reveals that Cyclosporine given to patients with AMI does not have better clinical outcome compared with placebo at improving LVEF, mortality, rehospitalisation, recurrent MI and heart failure occurrence. This study, however, verifies that Cyclosporine is well tolerated when administered in patients with AMI. The interpretation of these findings should take into consideration, the selection and performance bias across all studies. In addition to that, the low quality of included studies and the present of heterogeneity between studies may also have contributed to inaccurate results. More studies are needed in the future to examine the effectiveness of Cyclosporine in AMI, which should include the non-RCT articles and articles from other databases as well. Although cyclosporine holds limited promise as a cardioprotective agent for reducing reperfusion injury in STEMI, we should still uphold the potential of MPTP inhibitors in improving the clinical outcomes of reperfusion therapy. Perhaps, there is a need for the search of a novel cardioprotective agent or better strategies to limit reperfusion injury.FR, SA, WM, SS, TMK, and LT-HT: Perform the initial search and manuscript writing; data analysis was performed by FR, SA, WM, DB-CW, TMK, K-GC, C-FN, LM, L-HL, and B-HG: Have given critical comment on the final version. Manuscript was finalized by TMK, C-FN, LM, L-HL, and B-HG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetylgalactosaminyltransferase14 (GALNT14) has been proven to predict the progression-free survival (PFS), overall survival (OS) and response to chemotherapy in various types of gastrointestinal (GI) cancers. However, its role in PDA has not been studied. This study aims to investigate whether the GALNT14 SNP genotype can be a prognostic marker for PDA. A cohort of one hundred and three PDA patients having received surgical resection were retrospectively enrolled. GALNT14 genotypes and the clinicopathological parameters were correlated with postoperative prognosis. The genotype analysis revealed that 19.4%, 60.2% and 20.4% of patients had the GALNT14 “TT”, “TG” and “GG” genotypes, respectively. The patients with the “GG” genotype had a mean OS time of 37.1 months  and those with the “non-GG” genotype had a mean OS time of 16.1 months (95% CI: 13.1–19.2). Kaplan–Meier analysis showed that the “GG” genotype had a significantly better OS compared to the “non-GG” genotype (p = 0.005). However, there was no significant difference between the “GG” and “non-GG” genotypes in PFS (p = 0.172). The baseline characteristics between patients with the “GG” and “non-GG” genotypes were compared, and no significant difference was found. Univariate followed by multivariate Cox proportional hazard models demonstrated the GALNT14 “GG” genotype, negative resection margin, and locoregional disease as independent predictors for favorable OS . Sensitivity analysis was performed in each subgroup to examine the relationship of GALNT14 with different clinicopathological variables and no heterogeneity was found. The GALNT14 “GG” genotype is associated with favorable survival outcome, especially OS, in patients with resected PDA and could serve as a prognostic marker.Pancreatic ductal adenocarcinoma (PDA) is notorious for its poor prognosis. The current mainstay of treatment for PDA is surgical resection followed by adjuvant chemotherapy. However, it is difficult to predict the post-operative outcome because of the lack of reliable markers. The single-nucleotide polymorphism (SNP) of  Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer with poor prognosis, characterized by insidious clinical presentation and limited therapeutic options. Lack of validated screening and predictive biomarkers further complicates this condition . AlthougSo far, carbohydrate antigen 19-9 (CA19-9) remains the only U.S. Food and Drug Administration (FDA)-approved biomarker for PDA though some shortcomings still exist . ConsequGALNT14) was previously known as a gene encoding a catalytic enzyme for O-glycosylation of the death receptor (DR)-4 and 5 [rs9679162 of GALNT14 was first discovered correlating with the therapeutic outcome in patients with hepatocellular carcinoma [N-acetylgalactosaminyltransferase14 . Thirty-six patients were excluded because of the absence of major clinicopathological data on medical records. One hundred and three PDA patients were finally enrolled for baseline and OS analysis (Group 1). For PFS analysis, eighteen patients were excluded from group 1 because of either initial distant metastasis (n = 15) or the lack of following image study (n = 3). In total, eighty-five patients qualified for PFS analysis (Group 2).Surgical samples of 144 pancreatic tumor patients resected between year 1990 and 2010 were retrieved from a tertiary hospital’s tissue bank and sent for For patients’ clinical parameters, age, gender, initial tumor-node-metastasis (TNM) stage, laboratory tests, and adjuvant chemotherapy were collected. TNM stage was assessed according to American Joint Committee on Cancer (AJCC) 8th edition. Laboratory tests comprised total bilirubin, CA 19-9, and carcinoembryonic antigen (CEA). Intravenous and oral adjuvant chemotherapy records were both included. For pathological parameters, free resection margin, tumor size, tumor location, tumor grading, peritoneal implantation, lymphovascular permeation, and perineural invasion were recorded. OS was calculated from the date of operation to the date of death or last follow up. PFS was calculated from the date of operation to disease progression proven by image studies. This study was executed under the approval of the institutional review board of Chang Gung Memorial Hospital, Taiwan (107-2634C).GALNT14 GenotypingGALNT14 was conducted as described in previous literature [GALNT14 gene covering rs9679162. The SNP genotype was defined by direct sequencing after polymerase chain reaction amplification.Genotyping of terature . BrieflyStatistical Analysist-test or Mann–Whitney U test was used for continuous variables with or without normal distribution. Kaplan–Meier survival analysis with the log-rank test was used to compare outcomes. Clinicopathological parameters were analyzed to identify the predictive factors for OS and PFS by univariate and multivariate analysis with Cox proportional hazards regression model. Sensitivity analysis of the GALNT14 genotyping test was examined in different subgroups by the Cox model. The time points when patients were lost to follow up were treated as censored data. p < 0.05 indicated a statistically significant difference. All statistical analyses were performed with SPSS version 22.0 software  except the Cochran–Armitage trend test and Cochran’s Q test with R Core Team 2013 .The characteristics data were presented as ratios (%) for categorical variables, means ± standard deviation for continuous variables with normal distribution, and median (range) for continuous variables with a non-normal distribution. The normal and non-normal distribution were differentiated by the Kolmogorov–Smirnov method. For comparisons between groups, the Chi-square or Fisher’s exact tests were used for categorical data, and the two-sample Student’s GALNT14 “TT”, “TG” and “GG” genotypes, respectively. This genotype distribution did not deviate significantly from the previous three cohorts with other types of GI cancers  [The analysis of 103 PDA patients showed that 19.4%, 60.2% and 20.4% had the ctively) ,14,15. BGALNT14 genotype correlated with prognosis, we compared the survival between different genotypes. We found the patients with the “GG” genotype had a mean OS time of 37.1 months  and those with the “non-GG” genotype had a mean OS time of 16.1 months (95% CI: 13.1–19.2). On the other hand, the “GG” genotype had a mean PFS time of 29.4 months (95% CI: 9.7–49.2) and the “non-GG” genotype had 10.6 months (95% CI: 7.8–13.5). Kaplan–Meier analysis showed that the “GG” genotype had a significantly better OS compared to the “non-GG” genotype, with a distinguishable survival curve (p = 0.005). However, there was no significant difference between the “GG” and “non-GG” genotypes in PFS (p = 0.172). The baseline characteristics between patients with the “GG” and “non-GG” genotypes were compared, and no significant difference was found . In addition to the GALNT14 “GG” genotype, negative resection margin, no initial distant metastasis, and adjuvant chemotherapy also showed distinguishable survival curves on Kaplan–Meier survival plot . Stepwise multivariate analysis proved the GALNT14 “GG” genotype, negative resection margin, and no initial distant metastasis as independent predictors for favorable OS . The multivariable-adjusted hazard ratio (HR) of overall mortality was 0.273 (95% CI: 0.114–0.652) for the GALNT14 “GG” genotype compared to the “non-GG” genotype; 0.434 (95% CI: 0.198–0.949) for negative resection margin compared to positive margin; 0.302 (95% CI: 0.109–0.833) for local disease compared to initial distant metastasis . Univariate analysis revealed that only tumor size was borderline significantly associated with PFS (p = 0.055). The multivariable-adjusted HR of disease progression was 0.601 (95% CI: 0.357–1.011) for patients’ tumor size smaller than 10 cm3 compared with larger than 10 cm3 .In order to clarify the segregation of Kaplan–Meier curves for OS (and PFS) of the “GG” from the “non-GG” genotype occurring mainly after a follow up of one year A,F, we c2 = 0%, p = 0.9997).We also performed the sensitivity analysis by using Cox proportional hazard models in different subgroups. The result was shown on the forest plot . The hazGALNT14-rs9679162 in predicting therapeutic outcomes was initially established in advanced hepatocellular carcinoma (HCC) patients receiving chemotherapies [GALNT14-rs9679162. In other words, not only rs9679162, but also other variants located within a 5 kb intron region of GALNT14, centered by rs9679162 could serve as therapeutic outcome predictors. This finding suggested that patients carrying the “TT” genotype at rs9679162, actually harbor a 5 kb genomic region (around GALNT14-rs9679162), which is genetically different from those from the “non-TT” genotype. The sequence differences may thereby impact GALNT14 mRNA and protein expression, as variations in the intron region have been found to affect many aspects of regulatory events for gene expression, including transcription efficiencies and alternative splicing [GALNT14 are different between patients with the “TT” and “non-TT” genotypes. Among them, patients with the “TT” genotype were correlated to a lower level of GALNT14 in HCC tissues, while patients with the “GG” genotype were correlated to a higher level [The role of herapies ,12,16. Isplicing ,19,20,21er level .GALNT14 belongs to the GALNTs family, which is responsible for the initiation of O-linked glycosylation in protein by forming a structure called Tn antigen [GALNT14 expression has been demonstrated in numerous types of cancers, including breast cancer, ovarian cancer, pancreatic carcinoma, kidney cancer and HCC, although its growth regulatory role remains controversial in these cancers—some studies suggest an oncogene-like function, while others suggest a tumor suppressive role [ antigen . O-linkeive role ,25,26,27GALNT14-rs9679162 and chemotherapy treatment outcomes in HCC, the predictive role of GALNT14-rs9679162 has also been demonstrated in several other types of GI cancers, including cholangiocarcinoma, colorectal cancer, gastric signet ring cell cancer, and esophageal squamous cell cancer. Intriguingly, in only HCC and esophageal cancer (“carcinoma”), patients with the “TT” genotype had a better prognosis, while in the other cancers (“adenocarcinoma”), patients with the “GG” genotype had a better outcome [GALNT14, while the “GG”, a higher level, as observed in HCC [GALNT14 protein in PDA serves as a tumor suppressor, consistent with a previous report that GALNT14 sensitizes the TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death by glycosylating DR4 and DR5 [In addition to the correlation between  outcome ,14,15,16d in HCC , one can and DR5 . Convers and DR5 ,27. Acco and DR5  IntriguiGALNT14-rs9679162 “GG” genotype had a significantly lower hazard ratio compared to the “non-GG” genotype. After removing confounding factors by multivariate analysis, the “GG” genotype remained to be a favorable predictor. The Kaplan–Meier plot disclosed remarkably distinguishable survival curves between the “GG” and “non-GG” genotypes after the follow-up period of one year (p = 0.185) or on the Kaplan–Meier plot (p = 0.172). Nonetheless, differentiable survival curves could still be seen on the Kaplan–Meier plot after the follow-up period of one year  of “GG” patients have already died, the difference is apparently due to a subgroup of patients. To explore the causes of survival curve segregation appearing mainly after follow up of one year (OS and PFS), we analyzed whether different characteristics existed between patients who died or had disease progression before and after follow up of one year . The comThe literature revealed that adjuvant chemotherapy could benefit the survival of PDA after operation ,30,31. H3) was associated with a possibly better PFS in Kaplan–Meier analysis (p = 0.047) and univariate Cox model (p = 0.055). The insignificant result in the Cox model might be attributed to the inadequate patient numbers. Previous studies discovered that either short-term surgical outcome or postoperative long-term survival differs dramatically between patients with tumors more than and less than 2 cm in size [3). This was quite consistent with that in former studies, where the tumor size was calculated in cubic root (2.15 cm). It is noteworthy that CA19-9, which has been used as a prognostic marker of PDA, showed poor correlation with the outcomes in our survival analyses.Concerning the other clinicopathological variables, we found two independent parameters associated with significant OS differences including resection margin and initial metastases in univariate and multivariate Cox models. The Kaplan–Meier plot showed consistent results and distinguishable survival curves. Free resection margin had an impact on better survival in patients with PDA and was compatible with previous studies ,35. The  in size ,38,39. OGALNT14 genotype for prognosis and avoid bias, prospective studies may need to be performed and examine our findings in the future. Besides, though we collected patients from our facility in a 20 year period, the number of cases seemed inadequate to some extent. For example, the “GG” genotype had a trend of better PFS in survival analysis but did not reach statistical significance. In the Cox model and sensitivity analysis, some variables could not be compared due to the lack of events. Furthermore, the patients all belonged to a Mandarin population from a single medical center. Future large-scale and multicenter research with different ethnicities need to be conducted to verify our results. Lastly, a study with a head-to-head comparison between GALNT14 and CA19-9 as a promising prognostic biomarker for PDA needs to be designed.Admittedly, there were some limitations in our work. First, this was a retrospective cohort study with data retrieval from previous specimens and records. In order to understand more real applicability of the GALNT14-rs9679162 “GG” genotype was associated with favorable OS in patients with resected PDA and could be considered as a prognostic marker. Free resection margin and locoregional disease played a role in favorable OS as well. Tumor size might be a factor associated with patients’ PFS.In conclusion, the


The objective of this study was to investigate whether a soluble polymer and aldehyde-scavenger, polyvinylalcohol-carbazate (PVAC), can inhibit hemolysis in the storage of red blood cells (RBC).The effect of PVAC was assessed over a wide range of concentrations, using absorption spectroscopy to evaluate the level of hemolysis. Moreover, osmotic stability and aldehyde-scavenging potential of RBC were assessed after storage in PVAC.After test tube storage for two weeks, red blood cell hemolysis was lower with PVAC compared to controls . A higher level of hemolysis led to a pronounced effect with PVAC. RBC stored in PVAC improved both the binding of free aldehydes (p <0.001) and the osmotic stability (p = 0.0036).Erythrocytes stored with PVAC showed less hemolysis, which might be explained by the ability of PVACs to stabilize the cell membrane and decrease oxidative injury. Intrinsic causes are either structural or functional defects in the red blood cells (RBCs) which explains diseases such as thalassemia, sickle-cell disease or enzyme deficiencies. Extrinsic causes occur in the external RBC environment, e.g. autoimmune hemolytic anemia, where antibodies directed towards RBCs are formed or infections causing hemolytic uremic syndrome where toxins lyse RBCs Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript, “Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysis” assesses whether PVAC is capable of inhibiting hemolysis in stored red blood cell units. RBC units with PVAC exhibited higher aldehyde binding and osmotic stability, resulting in reduced hemolysis. The authors postulate that PVAC administration may prevent the propagation of damage by oxidative byproducts. Further, PVAC appears to have a membrane stabilizing effect per hypotonic studies in the manuscript. Given that no toxicity has been observed with PVAC, the aldehyde scavenger represents a promising additive agent for RBC storage.While the manuscript describes the methods in detail, the background could use more insight into current understanding of hemolysis during storage and relation to contemporary storage protocols. Further, the mechanism of carbonylation and any preventative pathways should be introduced. The methods section is thoroughly outlined, though the first section should be reworked to lay the foundation for later topics so that the section is easier to follow. The discussion ties together ideas and addresses any concerns with PVAC administration by citing toxicity data, though future studies will be necessary. Content feedback and grammatical edits may be found below.1. Content Feedback1.1. Page 3, Line 49: The terminology “such anti-hemolytic agents” should not be used here, unless these agents, or the notion thereof, are directly introduced in the preceding sentence.1.2. Page 3, Line 58: It is stated that hemolysis shortens the possible length of storage. This statement may be bolstered with specification of contemporary RBC storage parameters and how these are contingent upon in-bag hemolysis.1.3. Page 3, Line 59: How does carbonylation occur? Are there mechanisms in vivo that prevent carbonylation and/or its propagation?1.4. Page 5, Line 86: Please expand upon “desired buffer and concentration.” While covered later in the methods, expanding upon this (i.e. listing the buffer options) would make this section more understandable and enhance cohesiveness of the methods section as a whole. Components should at least be noted for frame of reference.1.5. Page 5, Line 87: The sentence states “the whole blood chamber model,” but this model has not yet been introduced. In the same vein of 1.4, adding detail here will help with understandability of the section as a whole.1.6. Page 5, Line 88: By which metrics was donor health assessed?1.7. Page 5, Line 95: Why was a second addition of PVAC employed for only the warm storage group, and not the cold storage group, following one week?1.8. Page 6, Line 109: Please specify which signature corresponds to 540 nm (Q-band) and, thus, why absorbance at this wavelength was us used in analyses.1.9. Page 8, Line 158: Please introduce what C3a is and why it is significant for assessment.1.10. Page 11, Line 226: What is DNR 2008/264?1.11. Page 13, Line 284: For figure four, the Y-axis reads “free aldehydes (%),” while the caption reads “free acetaldehyde.” Given that other aldehydes (i.e. MDA) may be present in the sample with storage age, perhaps the Y-axis should specify “acetaldehydes.”1.12. Page 14, Line 332: What additional stresses are present at room temperature to cause additional hemolysis? Please specify. This point may further be driven by introducing contemporary storage parameters and why cold storage is instituted  in the background section.2. Grammatical Edits2.1. Page 3, Line 44: Prior to using the acronym “RBCs” in the body text, please introduce this in parentheses following “red blood cells.” In this section, “RBCs” is used, but throughout the body “RBC” is later used to refer to plural erythrocytes. These uses need be altered to “RBCs” for clarity.2.2. Page 3, Line 46: Please change “haemolytic” to “hemolytic” for consistency with other uses of hemolysis throughout the manuscript.2.3. Page 3, Line 47: A comma needs to be added prior to “where toxins lyse the RBC” for readability.2.4. Page 3, Line 49: Please change “haemolytic” to “hemolytic” (see 2.2).2.5. Page 4, Line 82: Both uses in of “is” here should be changed to “are.”2.6. Page 6, Line 108: Minor typo of “minuntes.”2.7. Page 8, Line 172: Should “immune precipitation” read “immunoprecipitation” here?2.8. Page 12, Line 257: Please change the comma following “spectra” to a period.2.9. Page 13, Line 280: Please change the comma following “acetaldehyde” to a period.2.10. Page 13, Line 290: Please begin the sentence with “blood compatibility,” or some other terminology to complete this sentence.2.11. Suppl. Line 6: “a appropriate” should be changed to “an appropriate.”2.12. Suppl. Line 28: Please change “ice cold” to “ice-cold” for consistency with previous usage.2.13. Suppl. Line 119: “none-modified” should be change to “unmodified.”2.14. Suppl. Line 149: For consistency with other uses of “hem-“ in the main text, please change “haemoglobin” to “hemoglobin.”2.15. Suppl. Line 154: See 2.14.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  3 Nov 2019Response to reviewers1. Content Feedback1.1. Page 3, Line 49: The terminology “such anti-hemolytic agents” should not be used here, unless these agents, or the notion thereof, are directly introduced in the preceding sentence.We agree with the comment and have rephrased the statement accordingly. 1.2. Page 3, Line 58: It is stated that hemolysis shortens the possible length of storage. This statement may be bolstered with specification of contemporary RBC storage parameters and how these are contingent upon in-bag hemolysis.We agree that this would strengthen the introduction to our work. We have expanded the introduction to include more information about the storage lesion including the role of carbonylation and how it relates to contemporary RBC storage.1.3. Page 3, Line 59: How does carbonylation occur? Are there mechanisms in vivo that prevent carbonylation and/or its propagation?Please see answer to 1.2 1.4. Page 5, Line 86: Please expand upon “desired buffer and concentration.” While covered later in the methods, expanding upon this (i.e. listing the buffer options) would make this section more understandable and enhance cohesiveness of the methods section as a whole. Components should at least be noted for frame of reference.We appreciate the comment by the reviewer and we have revised accordingly. 1.5. Page 5, Line 87: The sentence states “the whole blood chamber model,” but this model has not yet been introduced. In the same vein of 1.4, adding detail here will help with understandability of the section as a whole.We agree that this sentence was out of context and it has been rephrased to reference biocompatibility. 1.6. Page 5, Line 88: By which metrics was donor health assessed?“Healthy donors” are assessed using a screening process after which their blood donations can be used for clinical transfusion. This is a rigorous process that includes medical and laboratory assessments that determine the health status of the donor and that follow internationally established standards. A brief statement has been added in the section on RBC preparation. 1.7. Page 5, Line 95: Why was a second addition of PVAC employed for only the warm storage group, and not the cold storage group, following one week?The rationale for multiple additions in the warm storage group is that we expected a greater degree of hemolysis during warm storage, which was based on knowledge from laboratory and clinical practices utilizing cold storage to limit hemolysis. The lowering of temperature leads to a slower? metabolism which lowers oxidative stress and lysis of RBC. This was further confirmed in our study where the increase in temperature increased the hemolysis. The materials and methods has been edited to explain the rationale. 1.8. Page 6, Line 109: Please specify which signature corresponds to 540 nm (Q-band) and, thus, why absorbance at this wavelength was used in analyses.Depending on the oxidative state of hemoglobin the absorbance spectrum changes. RBC stored in the presence of oxygen are generally in the state of oxyhemoglobin which has an absorbance peak of approximately 540 nm. We confirmed this in our study by measuring the absorbance spectrum of hemoglobin between 300 and 700 nm . We subsequently determined that 540 nm was an appropriate wavelength for detection also in our investigations. In materials and methods we have added a paragraph to clarify the wavelengths at which hemoglobin was measured. 1.9. Page 8, Line 158: Please introduce what C3a is and why it is significant for assessment.In materials and methods we have added a description of C3a, which is a molecule formed in the complement cascade that signals inflammation. In the whole blood model, increasing levels of C3a is an appropriate marker for general complement activation. 1.10. Page 11, Line 226: What is DNR 2008/264?This is an identification number used by the Swedish Regional Ethical Review Board. We have clarified this in our ethical statement in the materials and methods and added the statement to our supporting information. 1.11. Page 13, Line 284: For figure four, the Y-axis reads “free aldehydes (%),” while the caption reads “free acetaldehyde.” Given that other aldehydes (i.e. MDA) may be present in the sample with storage age, perhaps the Y-axis should specify “acetaldehydes.”We appreciate this astute observation and have clarified that we are indeed measuring not only acetaldehyde but in fact all free aldehydes. The method used relies on the enzyme acetaldehyde dehydrogenase and measures reduction of the substrates for the enzyme; we have also tested and confirmed that the method works with for example MDA. 1.12. Page 14, Line 332: What additional stresses are present at room temperature to cause additional hemolysis? Please specify. This point may further be driven by introducing contemporary storage parameters and why cold storage is instituted  in the background section.We have added additional information in the introduction/background regarding contemporary storage parameters. Please also see our response to point 1.2.2. Grammatical Edits2.1. Page 3, Line 44: Prior to using the acronym “RBCs” in the body text, please introduce this in parentheses following “red blood cells.” In this section, “RBCs” is used, but throughout the body “RBC” is later used to refer to plural erythrocytes. These uses need be altered to “RBCs” for clarity.We have changed the abbreviation so that only RBCs is used throughout the manuscript, since we only refer to red blood cells in the plural form. 2.2. Page 3, Line 46: Please change “haemolytic” to “hemolytic” for consistency with other uses of hemolysis throughout the manuscript. 2.3. Page 3, Line 47: A comma needs to be added prior to “where toxins lyse the RBC” for readability.2.4. Page 3, Line 49: Please change “haemolytic” to “hemolytic” (see 2.2).2.5. Page 4, Line 82: Both uses in of “is” here should be changed to “are.”2.6. Page 6, Line 108: Minor typo of “minuntes.”2.7. Page 8, Line 172: Should “immune precipitation” read “immunoprecipitation” here?2.8. Page 12, Line 257: Please change the comma following “spectra” to a period.2.9. Page 13, Line 280: Please change the comma following “acetaldehyde” to a period.2.10. Page 13, Line 290: Please begin the sentence with “blood compatibility,” or some other terminology to complete this sentence.2.11. Suppl. Line 6: “a appropriate” should be changed to “an appropriate.”2.12. Suppl. Line 28: Please change “ice cold” to “ice-cold” for consistency with previous usage.2.13. Suppl. Line 119: “none-modified” should be change to “unmodified.”2.14. Suppl. Line 149: For consistency with other uses of “hem-“ in the main text, please change “haemoglobin” to “hemoglobin.”2.15. Suppl. Line 154: See 2.14.All grammatical edits (2.2 – 2.15) suggested by the reviewer have been corrected. Furthermore, an experienced English proof reader has reviewed the manuscript and made additional edits for readability.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 13 Nov 2019Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysisPONE-D-19-23341R1Dear Dr. Sellberg,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Jeffrey Chalmers, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 22 Nov 2019PONE-D-19-23341R1 Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysis Dear Dr. Sellberg:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Jeffrey Chalmers Academic EditorPLOS ONE