In a previous study, we utilized a proteomic approach and found a significant reduction in phosphatidylethanolamine-binding protein 1 (PEBP1) protein level in the spinal cord at 3 h after ischemia. In the present study, we investigated the role of PEBP1 against oxidative stress in NSC34 cells in vitro, and ischemic damage in the rabbit spinal cord in vivo. We generated a PEP-1-PEBP1 fusion protein to facilitate the penetration of blood-brain barrier and intracellular delivery of PEBP1 protein. Treatment with PEP-1-PEBP1 significantly decreased cell death and the induction of oxidative stress in NSC34 cells. Furthermore, administering PEP-1-PEBP1 did not show any significant side effects immediately before and after ischemia/reperfusion. Administration of PEP-PEBP1 improved the Tarlov’s neurological score at 24 and 72 h after ischemia, and significantly improved neuronal survival at 72 h after ischemia based on neuronal nuclei (NeuN) immunohistochemistry, Flouro-Jade B staining, and western blot study for cleaved caspase 3. PEP-1-PEBP1 administration decreased oxidative stress based on malondialdehyde level, advanced oxidation protein products, and 8-iso-prostaglandin F2α in the spinal cord. In addition, inflammation based on myeloperoxidase level, tumor necrosis factor-α level, and high mobility group box 1 level was decreased by PEP-1-PEBP1 treatment at 72 h after ischemia. Thus, PEP-1-PEBP1 treatment, which decreases oxidative stress, inflammatory cytokines, and neuronal death, may be an effective therapeutic strategy for spinal cord ischemia. Interruption of the aorta or its segmental arteries results devastating and unpredictable complications, such as paraplegia caused by ischemia in spinal cord  during e2O2-induced oxidative stress in NSC34 cells in vitro and ischemic damage in the rabbit spinal cord in vivo.In the previous study, we generated a PEP-1-PEBP1 fusion protein and confirmed it could penetrate the blood-brain barrier and plasma membrane of neurons . In the NSC34 cells were used in the present study and cells were maintained in the conditions described in the previous study . SpecialXhoI restriction site, and the PEBP1-specific anti-sense primer, 5′-GGA TCC CTA CTT CCC AGA CAG C-3′, which contained a BamHI restriction site. PEP-1-PEBP1 was by ligation of a TA cloning vector with human PEBP1 cDNA and expression plasmid was inserted into a pET-15 vector with His-PEP-1-PEBP1 or His-PEBP1. PEP-1-PEBP1 and control-PEBP1 proteins were produced as described in a previous study [The cDNA sequence for human PEBP1 was amplified by polymerase chain reaction (PCR) using the PEBP1-specific sense primer 5′-CTC GAG ATG CCG GTG GAC C-3′, which contained an us study , and theus study ,18. Transduction of PEP-1-PEBP1 proteins into NSC34 cells was confirmed as described previously . BrieflyIntracellular delivery of PEP-1-PEBP1 or control-PEBP1 protein was confirmed by immunocytochemical staining for polyhistidine, as described previously ,16. Brie2O2) for 3 h and 10 min for TUNEL and DCF-DA staining as described previously [To elucidate the effects of PEP-1-PEBP1 or control-PEBP1 protein on DNA damage and oxidative stress, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining was done as described in previous studies ,16. Brieeviously , respecteviously ,20.2O2 for 5 h. Cell viability was measured at 450 nm using an ELISA microplate reader , and cell viability was expressed as a percentage value versus that in the untreated control cells.Effects of PEP-1-PEBP1 or control-PEBP1 protein on cell viability was assessed based on a WST-1 assay kit  as described previously . BrieflyEqual amounts of cell lysates were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in TBST buffer . Then, the membrane was probed with the rabbit anti-caspase 3 , and rabbit anti-cleaved caspase 3 , and horse anti-rabbit IgG . The proteins were detected using enhanced chemiluminescent  protocol according to the manufacturer’s instructions .Male New Zealand white rabbits (1.2–1.5 kg) were purchased from Experimental Animal Center  and were housed as described in the previous studies ,21. The To observe dose-dependent effects of PEP-1-PEBP1 on motor neurons in ventral horn of spinal cord after ischemia, the animals were divided into five groups: Sham-operated (control), PEP-1 peptide, 10 mg/kg control-PEBP1, 1 mg/kg PEP-1-PEBP1, 3 mg/kg PEP-1-PEBP1, and 10 mg/kg PEP-1-PEBP1 treated groups. Animals received treatment of PEP-1 peptide, control-PEBP1, or PEP-1-PEBP1 immediately after reperfusion.The ischemic surgery was performed as described in a previous study . Briefly2 and PaCO2) before ischemic surgery and 10 min after reperfusion, as described previously [Blood in all groups was obtained from ear vein to measure the blood glucose, pH, and arterial blood gases  were anesthetized with 2 g/kg urethane (Sigma) after the neurological assessment and perfused transcardially, as described previously [5-L6) of spinal cord were removed and 30-μm-thick sections were obtained using a cryostat .Rabbits  was conducted as described previously ,21. Sect®, CyberMetrics, Scottsdale, AZ, USA) as described previously [The number of NeuN-immunoreactive cells in all the groups were counted using an image analysis system  histofluorescence staining was conducted according to the method published by Candelario-Jalil et al. . In brien = 5 in each group) were euthanized with overdose of urethane (Sigma) 72 h after reperfusion, and spinal cord tissue at L5-L6 levels were obtained. Quantitative analysis was conducted by western blot analysis for caspase 3 and c-caspase 3 in the spinal cord. Briefly, animals were sacrificed using 2 g/kg of the anesthetic urethane (Sigma-Aldrich). Lumbar segments (L5-L6) of spinal cord were removed and used for western blot study as described in a previous study [To measure biochemical parameters in spinal cord tissue, control, PEP-1 peptide-treated, 10 mg/kg Control-PEBP1-treated, and 3 mg/kg PEP-1-PEBP1-treated rabbits , MPO , HMGB , TNF-α , and 8-iso-PGF2α  levels were measured by commercially available ELISA kits. AOPP levels were measured by a spectrophotometric method (Schimadzu UV 1601 spectrophotometer) in the presence of potassium iodide at 340 nm as demonstrated by Witko-Sarsat et al.  and calit-test or a one-way analysis of variance (ANOVA), followed by Bonferroni’s post-hoc test using GraphPad Prism 5.01 software .Data were shown as mean with standard errors of mean or 95% confidence interval and analyzed statistically using by Student PEBP1 gene and a PEP-1 expression vector (b+- → Ni2+- nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography. Western blot analysis with a polyhistidine antibody identified PEP-1-PEBP1 and control-PEBP1 proteins at approximately 23 kDa and 25 kDa, confirming the successful expression of these proteins (PEP-1-PEBP1 was manufactured by fusion of human n vector A. Followproteins B.Concentration- and time-dependent changes in polyhistidine expression were determined in NSC-34 cells. Weak polyhistidine expression was found at 1 μM PEP-1-PEBP1 treatment and expression increased dose-dependently by 3 μM PEP-1-PEBP1 treatment. However, polyhistidine expression was not observed upon treatment with any concentration of control-PEBP1 A. In addThe intracellular stability of transduced PEBP1 protein in NSC34 cells was assessed by western blotting for polyhistidine. Polyhistidine protein levels had decreased 24 h after PEP-1-PEBP1 treatment, and had disappeared at 36 h after treatment C. Transduced PEBP1 was observed by polyhistidine immunocytochemistry, and no signals were observed in the control NSC34 cells or cells 1 h after 3 μM control-PEBP1 treatment. In contrast, treatment with 3 μM PEP-1-PEBP1 showed polyhistidine immunoreactivity in the cytoplasm and cell membrane, but not in the nucleus of NSC34 cells D.2O2 for 3 h significantly decreased cell viability to 47.96% of the viability of the control group. However, pre-treatment with PEP-1-PEBP1 protein for 1 h before exposure to H2O2 showed dose-dependent increases in cell viability, and 3 μM PEP-1-PEBP1 showed significant increases in cell viability compared to that in the control-PEBP1-treated group, and cell viability was 66.37% of the viability of the control group (Exposure to 1 mM Hol group A. Howeve2O2 for 3 h showed strongly DCF-DA positive cells, and reactive fluorescence intensity was significantly increased compared to that in the control group. Pretreatment with control-PEBP1 did not significantly change DCF-DA reactive fluorescence in NSC34 cells compared to that in the H2O2-treated vehicle group. However, PEP-1-PEBP1 pretreatment significantly decreased DCF-DA reactive fluorescence compared to that in the H2O2-treated vehicle group (ROS formation was measured by DCF-DA-fluorescent staining in NSC34 cells. Treatment with 1 mM Hle group B.2O2 in NSC34 cells. Exposure to 1 mM H2O2 for 5 h showed abundant TUNEL-positive cells, and reactive fluorescence intensity was significantly increased compared to that in the control group. Pretreatment with PEP-1-PEBP1 before exposure to 1 mM H2O2 significantly decreased the number of TUNEL-positive cells compared to that in the H2O2-treated vehicle group, while pretreatment with control-PEBP1 showed a similar number of TUNEL-positive cells with that in the H2O2-treated vehicle group (Apoptotic cell death was assessed by TUNEL staining 5 h after exposure to Hle group C.2O2 in NSC34 cells by western blot analysis for caspase 3 and c-caspase 3. Caspase 3 protein levels did not show any significant changes in all groups after control-PEBP1 or PEP-1-PEBP1 treatment, while c-caspase 3 protein levels were dramatically increased in vehicle group after H2O2 exposure compared to that in the control group. Treatment with control-PEBP1 had no effects on the c-caspase 3 levels in NSC34 cells compared to that in the vehicle group, but PEP-1-PEBP1 treatment significantly reduced the c-caspase 3 protein levels by 46.98% of vehicle group in NSC34 cells (Cell death was also confirmed 5 h after exposure to H34 cells D.2, pCO2, and glucose levels, as well as distal MAP, were measured before ischemic surgery and 10 min after reperfusion. Administration of control-PEBP1 or PEP-1-PEBP1 did not significantly change these physiological parameters before ischemic surgery and 10 min after reperfusion (The acute side effects of control-PEBP1 or PEP-1-PEBP1 treatment on physiological conditions such as blood pH, pOerfusion . Effectserfusion .The effects of control-PEBP1 or PEP-1-PEBP1 treatment on neuronal cell death were evaluated by FJB staining and western blot for caspase 3 and c-caspase 3 in the spinal cord. In control group, FJB positive cells were not detected in the ventral horn of spinal cord. In the PEP-1 peptide- and control-PEBP1-treated groups, FJB positive cells were found in the ventral horn of spinal cord and c-caspase 3 was prominently increased compared to that in the control group. In these groups, there were significant differences on FJB positive cells and c-caspase levels in the spinal cord. In the PEP-1-PEBP1-treated group, fewer FJB positive cells were found in the spinal cord compared to that in the PEP-1 peptide- or control-PEBP-1-treated group, respectively. In addition, c-caspase 3 levels were also significantly decreased in the PEP-1-PEBP1-treated group compared to that in the PEP-1 peptide-treated group . In spinal cord tissues, levels of MDA, AOPP, and 8-iso-PGF2α, three oxidative stress biomarkers, were measured to determine ischemia-related oxidative stress in the spinal cord 72 h after ischemia. In the PEP-1 peptide-treated group, MDA, AOPP, and 8-iso-PGF2α levels were significantly increased in the spinal cord after ischemia by 225.04%, 145.64%, and 1049.17% of that of the control group, respectively. In the control-PEBP1-treated group, MDA and AOPP levels were similarly detected compared to those in the PEP-1 peptide-treated group, although 8-iso-PGF2α level was slightly decreased in the control-PEBP1-treated group. However, the administration of PEP-1-PEBP1 significantly reduced ischemia-induced elevations of MDA, AOPP, and 8-iso-PGF2α levels to 126.43%, 103.77%, and 402.18% of that of the control group, respectively . In the PEP-1 peptide-treated and control-PEBP1-trateed groups, MPO, TNF-α, and HMGB levels were significantly robustly increased in the spinal cord 72 h after ischemia compared to those in the control group. In this group, MPO, TNF-α, and HMGB levels in the spinal cord homogenates were 284.97%, 949.70%, and 471.92% of those in the control group, respectively. In the control-PEBP1-treated group, MPO level was similar to that in the PEP-1 peptide-treated groups, while TNF-α and HMGB levels were slightly decreased in the control-PEBP1-treated group compared to that in the PEP-1 peptide-treated group, respectively. Administration of PEP-1-PEBP1 ameliorated these dramatic increases of MPO, TNF-α, and HMGB levels in the spinal cord to 188.10%, 456.68%, and 203.31% of that of the control group, respectively . PEBP1 is involved in neuronal cell death and cognitive deficits in neurodegenerative diseases . In addiIn the present study, we observed concentration- and time-dependent delivery of PEP-1-PEBP1 proteins in the NSC34 cells, which have similar morphological and physiological properties as motor neurons ,34,35. H2O2-induced oxidative damage in NSC34 cells because glutamate-induced excitotoxicity has no specific effects in NSC34 cells [2O2 based on caspase 3 western blot, TUNEL, DCF-DA staining, and WST-1 assays. This result suggests that PEP-1-PEBP1 prevents neuronal death from oxidative damage via reducing ROS formation. Depletion of PEBP1 significantly increases generation of mitochondrial superoxide in HEK-499 cells [In this study, we observed the effects of control-PEBP1 and PEP-1-PEBP1 on H34 cells . Treatme99 cells . In cont99 cells  and ROS 99 cells .2, pCO2, or glucose levels. Spinal cord ischemia showed neurological deficits in rabbits’ hindlimb functions and posture judging from Tarlov’s criteria at 24 h and 72 h after ischemia/reperfusion. Administering control-PEBP1 resulted in similar neurological deficits in rabbits compared to that in the PEP-1 peptide-treated group at 24 h and 72 h after ischemia/reperfusion. In addition, NeuN-immunoreactive surviving neurons were few in the PEP-1-peptide or control-PEBP1 treated groups compared to that in the control group at 72 h after ischemia/reperfusion, while FJB positive cells and c-caspase 3 protein levels were significantly increased compared to that in the control group. However, the administration of PEP-1-PEBP1 showed dose-dependent improvements in rabbits’ neurological scores at 24 h and 72 h after ischemia/reperfusion, although the neurological scores were slightly decreased at 72 h after ischemia. In addition, administration of PEP-1-PEBP1 significantly ameliorated the increases of FJB positive cells and c-caspase expression in the spinal cord induced transient ischemia. Spinal cord ischemia is caused by aortal occlusion during aortic repair surgery [We examined the neuroprotective effects of control-PEBP1 or PEP-1-PEBP1 against ischemic damage in the rabbit spinal cord. Acute side effects of control-PEBP1 or PEP-1-PEBP1 10 min before and after ischemia were monitored, but we did not observe any significant change in physiological parameters such as pH, pO surgery . Depleti surgery ,14. In a surgery . This re surgery ,28. 2α has been considered a specific and sensitive indicator for peroxidation caused by ROS [2α levels to observe ischemia-induced lipid peroxidation and protein oxidation in the spinal cord. Spinal cord ischemia significantly elevated the MDA, AOPP, and 8-iso-PGF2α levels in the spinal cord at 72 h after ischemia/reperfusion, consistent with results of a previous study [2α levels in the spinal cord at 72 h after ischemia compared to that in the PEP-1 peptide-treated group. In contrast, PEP-1-PEBP1 significantly ameliorated increases of MDA, AOPP, and 8-iso-PGF2α levels in the spinal cord following ischemia, suggesting that administering PEP-1-PEBP1 reduces ischemia-induced lipid peroxidation and protein oxidation in the spinal cord.Spinal cord membranes consist of polyunsaturated fatty acids, and ischemia-induced ROS causes lipid peroxidation with MDA as a decomposition product . AOPP isd by ROS . In the us study . In the MPO is a hemoprotein expressed by polymorphonuclear neutrophils and is an indicator of neutrophil infiltration into tissues . In the 2O2-induced oxidative stress in NSC34 cells as well as ischemia-induced neuronal death in rabbit spinal cord, reducing inflammation and oxidative damage. These results suggest that the PEP-1-PEBP1 fusion protein may be applicable to reduce neuronal damage during aortic repair surgery and en bloc spondylectomy by suppressing oxidative stress and inflammation.In conclusion, we generated a PEP-1-PEBP1 fusion protein and observed the effective penetration of PEP-1-PEBP1 into NSC34 cells. In addition, we observed the neuroprotective effects of PEP-1-PEBP1 against H


Approximately 12-18% of family caregivers to older adults in the U.S. are 18-25 years old , yet minimal research has focused on this subgroup of caregivers . Individuals’ perceptions of an older adult’s social role relate to their attitudes toward older adults as a group . However, whether perceptions that emerging adult caregivers hold of older adults are specific to the social role of “care-recipient” has not been studied. A sample of 210 informal caregivers (ages 18-25) were surveyed to collect qualitative responses regarding perceptions of an older adult care-recipient (age 65+) and to assess quality of contact with the care-recipient and ageist attitudes. Participants were asked to provide five adjectives describing their older adult care-recipient. Approximately 43% provided a set of adjectives in which 80%-100% were coded as positive adjectives ; similarly, half of the sample’s adjective sets contained 0%-25% negative adjectives . The quality of contact with the care-recipient was significantly correlated (p<.001) with the percentage of positive (r=.47) and negative (r=-.49) adjectives. Scores on the Fraboni Scale of Ageism were also significantly correlated (p<.01) with the percentage of positive (r=-.19) and negative (r=.20) adjectives. Overall, these emerging adult caregivers had generally positive perceptions of their older adult care-recipients, and these perceptions reflected the positive quality of contact with the care-recipient. Less ageist attitudes’ relationship with more positive and less negative perceptions may have implications for experiences within a caregiving dyad.


More than 4 billion inhabitants in Asia depend on rice for 35–60% of the calories consumed in their diets, but new rice cultivars frequently do not reach expected yields because of poor rice grain filling. Here, we quantified the activities of enzymes involved in starch metabolization in rice to investigate the mechanisms regulating the accumulation and translocation of stem non-structural carbohydrates (NSC) under different levels of nitrogen fertilizer application. A pot experiment was conducted using two rice cultivars, Liangyoupeijiu (LYPJ) and Shanyou63 (SY63), under high and low nitrogen applications. Compared with high nitrogen application (HN), low nitrogen application (LN) increased stem NSC concentration before the heading stage and NSC translocation during the grain filling stage; concomitantly, LN significantly shortened the active grain filling period and increased the grain filling rate in superior spikelets. Compared with the LYPJ cultivar, SY63 exhibited a higher grain weight, higher grain filling percentage, and higher stem NSC concentration before heading and greater NSC translocation after heading. During the period between panicle initiation and heading, the activities of adenosine diphosphate-glucose pyrophosphorylase (AGP), starch synthase (StS), and starch branching enzyme (SBE), all enzymes involved in starch synthesis, increased under the LN treatment and positively correlated with increases in stem NSC. During grain filling, the activities of enzymes involved in starch-to-sucrose conversion  increased under the LN treatment and positively correlated with stem NSC remobilization. Overall, the investigated enzymes exhibited higher activities in SY63 than in LYPJ. Our results suggest that low nitrogen increases the activities of AGP, StS, SBE, α-amylase, β-amylase, and SPS, leading to increased accumulation and remobilization of stem starch and NSC in SY63. We conclude that calculated reductions in nitrogen application and the choice of an appropriate cultivar may improve rice grain yields via enhanced stem NSC accumulation and translocation, thereby reducing the costs and increasing the sustainability of rice production. Oryza sativa L.) provides 35–60% of the calories consumed by the more than 4 billion inhabitants of Asia  and Shanyou63 , were used in a pot experiment at Huazhong Agricultural University, Wuhan, China. LYPJ and SY63 were two elite rice hybrids that were widely planted in China  and 2.6 g N pot-1 (HN) in the form of urea, which were equal to N application rates of 21 kg ha-1 and 210 kg ha-1 in farmer field, respectively. Each N treatment and each cultivar was repeated three times. The nitrogen fertilizer applications were split-applied at a ratio of 4:3:3 at three different times: basal (1 day before transplanting), tillering stage (approximately 36 days after germination), and at panicle initiation (approximately 66 days after germination). Phosphorous (P) and potassium (K) in the form of KH2PO4 were applied as basal fertilizers at rates of 1.50 and 1.89 g pot-1, respectively. Basal fertilizers were applied by thorough mixing into the soil before transplanting; subsequent fertilizers were applied in a top-dressed manner by dissolving them in water and then irrigating. Eighteen pots were used for each of the four treatments testing rice variety and N level, for a total of 72 pots. During the entire experiment, plants received well water, maintaining a water depth of at least 2 cm. Pests and diseases were controlled using chemical pesticides.Germinated seeds were seeded to nursery plates containing sandy loam soil on 23 May 2015. On 17 June 2015, three uniform, 25-day-old seedlings were transplanted into each of 72, 12.0 L pots  filled with 10.0 kg soil. The soil used was a clay type soil and had the following properties: pH 7.1, 7.0 g organic matter kgUpon panicle initiation  when the average length of young panicles was about 1 mm , several main and large stems were chosen and tagged for each treatment. Panicles from main or large stems that flowered on the same day were chosen and tagged on the heading date . Six tagged main or large stems (culms and sheaths) from six plants in each treatment were sampled at 5-day or 6-day intervals from panicle initiation to maturity. Considering the effect of plant number on available N for each plant and experiment scale, only two large stems were tagged from each plant from the same pot for sampling, one for each time point. After the two samplings, the plants were not used for any other sampling.Three sampled stems were immediately frozen in liquid nitrogen and then stored at -80°C for determination of enzyme activity. The other stems were oven-dried at 80°C to constant weight and ground for the measurement of soluble sugars, starch, and sucrose. In total, we collected six samplings for measuring enzyme activity during the PI stage  and six samplings for measuring enzyme activity between heading and maturity .-1 d-1) was calculated between t1 and t2  was the total dry weight of the aboveground plant parts, grain yield (g plant-1) was the weight of full-filled grains, and harvest index (%) was calculated as the ratio of the grain yield to the biomass.At both heading and maturity, three entire plants per treatment were sampled and separated into leaves, stems (culms and sheaths), and panicles. All spikelets of the panicles were hand-threshed at maturity. Filled grains and unfilled grains were divided by submersing them in tap water. Empty grains were separated from unfilled grains using a seed winnowing cleanliness instrument. The dry weights of all parts, including leaves, stems, rachises, and spikelets were weighed after being oven-dried at 80°C to a constant weight. The numbers of filled, partially filled, and empty grains were counted, and the 1000-grain weight (g) and percentages of half-filled and fully-filled grains (%) were calculated. Biomass . After comparison to a standard curve of glucose, concentrations of soluble sugars and starch were calculated as mg glucose gg by 0.9 .-1 NaOH solution was added to 0.4 mL of filtered solution, then the mixture was boiled at 100°C for 5 min. After cooling, 2.8 mL of 9 mol L-1 HCl and 0.8 mL of 0.1% resorcinol were added and the reaction mixture was incubated at 80°C for 10 min, followed by determination of absorbance at 480 nm. After comparison to a standard curve of sucrose, the sucrose concentration was calculated as mg g-1 dry weight.Sucrose was determined according to the method of -1 dry weight) of a given plant part is the sum of concentrations of soluble sugars and starch. The total mass of NSC stored in stems  was calculated as stem biomass multiplied by NSC concentration. The apparent transferred mass of NSC from stems to grains during grain filling  is the TM at heading minus TM at maturity. The apparent ratio of transferred NSC from stems to grains  is the ratio of ATM to TM at heading. The apparent contribution of transferred NSC to grain yield  is the ratio of ATM to grain yield. In this article, NSC translocation was described by ATM, AR, AC, and δCNSC (defined as the differences in NSC concentrations at heading and at maturity).The NSC concentration , 2 mM ethylene diamine tetraacetic aicd (EDTA), 12.5% (v/v) glycerol, and 5% (w/v) polyvinylpolypyrrolidone (PVPP). The homogenate was centrifuged at 4°C and 12 000 rpm for 20 min and the supernatant was used in an enzyme extraction to determine the activities of AGP, StS, and SBE.Between the panicle initiation and heading stages, the three tagged stems were sampled every 5 days to measure the activities of three key enzymes involved in starch synthesis . The sampled stems were ground and extracted with a medium composed of 100 mM Hepes-NaOH (pH 7.6), 8 mM MgCl2, and 4 mM DTT. After incubating at 30°C for 15 min, the reaction was terminated by placing the tube into boiling water for 30 s. The resulting solution was centrifuged at 12,000 rpm for 10 min. Then, 100 μL of the supernatant was mixed with 3 μL of 10 mM nicotinamide adenine dinucleotide phosphate (NADP). The absorbance at 340 nm was measured after addition of phosphoglucomutase (0.4 unit) and glucose-6-P dehydrogenase (0.35 unit). The protein content of the enzyme extract was measured according to -1 protein h-1.The activity of AGP was determined according to 2, and pyruvate kinase (1.2 unit). After incubating at 30°C for 15 min, the solution was heated in boiling water for 30 s to terminate the reaction and centrifuged at 4°C and 12 000 rpm. Next, 60 μL of supernatant was mixed with 43 μL of 50 mM Hepes-NaOH (pH7.4), 10 mM glucose, 20 mM MgCl2, and 2 mM NADP in a tube and incubated for 10 min at 30°C. The absorbance was measured at 340 nm after the addition of hexokinase (1.4 unit) and glucose-6-P dehydrogenase (0.35 unit). The activity of StS is expressed as μmol NADPH mg-1 protein h-1.Starch synthase activity was measured according to 2-1% KI after cooling. The absorbance was measured at 610 nm and the remaining starch content was determined by comparing it to the starch standard. The activity of SBE is expressed as mg starch mg-1 protein h-1.Activity of SBE was determined according to Between the heading and maturity stages, we sampled stems every 6 days to determine the activities of key enzymes involved in starch-to-sucrose conversion .2-1% KI was measured at 610 nm. A control sample was prepared using deactivated enzyme boiled for 30 s. For the assay of β-amylase, the assay mixture consisted of 200 mM sodium acetate containing 0.1 mM EDTA (pH 3.6), 0.3 mL 0.067% starch solution, and 0.1 mL enzyme extract. The procedure was the same as for the α-amylase assay. The activities of α-amylase and β-amylase were expressed as mg starch hydrolyzed mg-1 protein h-1.For extraction of α-amylase and β-amylase, frozen stems were ground in pre-cooled 100 mM phosphate buffer (pH 6.5) and centrifuged at 4°C and 12,000 rpm for 20 min. The enzyme activities were determined as described by 2, 1 mM EDTA, 5 mM DTT, 1 mM phenazine methosulfate (PMSF), 1 mM benzamidine, and 3% (w/v) PVPP. After centrifuging at 4°C and 12,000 rpm for 20 min, the supernatant was used for the assay of SPS and SSs. For SPS, 30 μL enzyme preparation was added to a tube of 70 μL reaction medium containing 50 mM Hepes-NaOH (pH 7.5), 3.5 mm UDP-glucose, 3.5 mM fructose-6-P, and 10 mM MgCl2. The mixture was then incubated at 30°C and the reaction was terminated after 15 min by adding 100 μL 1.0 mol L-1 NaOH. Unreacted fructose-6-P was destroyed by placing the tubes in boiling water for 10 min. After cooling, 0.75 mL of 9 mol L-1 HCl and 0.25 mL of 0.1% (w/v) resorcinol were added and the tube was incubated at 80°C for 10 min. Absorbance was measured at 480 nm after cooling. For SSs, the reaction mixture contained 30 μL of enzyme preparation, 30 μL of 100 mM fructose, and 70 μL of medium containing 100 mM Hepes-NaOH (pH 8.5), 5 mM KCl, 5 mM NaCl, and 8 mM UDP-glucose. The reaction mixture was then incubated at 30°C for 15 min. The remaining steps were the same as for SPS determination. The enzyme activity of SPS and SSs were expressed as μmol sucrose synthesized mg-1 protein h-1.Extraction and assay of SPS and SSs were performed according to -1 protein h-1.The assays for AI, NI, and sucrose synthase activity in the cleavage direction (SSc) followed the protocol described by n = 4 for each cultivar, n = 8 for each nitrogen treatment). To determine the relations of enzyme activities with NSC translocation during the rapid decrease in stem starch and NSC 6–24 days after heading, we correlated enzyme activities involved in starch/NSC decreases using the differences between starch/NSC concentration at heading and days 6, 12, 18, 24 days after heading . The correlation analyses were performed based on a small number of samples in previous studies  test at a 5% probability level using the Statistix 9 software package . Linear correlation analysis was used to evaluate the relationships of enzyme activities with stem NSC accumulation and translocation using the SigmaPlot 10.0 software package . To determine the relations of enzyme activities with NSC accumulation during the rapid accumulation of stem starch and NSC 0–20 days after panicle initiation, we correlated enzyme activities with starch/NSC accumulation using the differences between starch/NSC concentrations on the date of panicle initiation and days 5, 10, 15, and 20 afterward .Under the LN application condition, grain filling in the superior spikelets of LYPJ and SY63 was completed approximately 19 days after heading. Under the HN application condition, this process was completed around day 23. No difference was observed in the inferior spikelets of LYPJ and SY63 between the LN and HN applications . Grain filling rate of superior spikelets increased by 16.7 and 26.4% in LYPJ and SY63, respectively , and the active grain filling period of superior spikelets shortened by 3.5 and 4.2 days in LYPJ and SY63, respectively . However, no significant differences were observed in active grain filling rate and grain filling period of inferior spikelets between LN and HN .The LN treatment significantly increased the grain filling rate and shortened the active grain filling period of superior spikelets compared with the HN treatment . The active grain filling period of SY63 superior spikelets was 1 day less than that in LYPJ under LN, but no difference was observed under HN . No significant differences were observed in the active grain filling period and grain filling rate of inferior spikelets of the LYPJ and SY63 cultivars .The superior spikelets of SY63 exhibited a higher grain filling rate than the superior spikelets of LYPJ by 17.4 and 8.4% under the LN and HN treatments, respectively, but a significant difference was only observed under the LN treatment . Low nitrogen increased the harvest index of LYPJ and SY63 , while LN decreased the half-filled grain percentage by 14.4 and 16.6% in LYPJ and SY63, respectively; a significant difference was observed only in SY63 . Grain weight was not influenced by nitrogen treatment . Plant biomass and grain yield were significantly decreased under the LN treatment in both LYPJ and SY63 compared with the HN treatment .The LN treatment increased the grain filling percentage of LYPJ and SY63, when compared with the HN treatment, but the differences were not significant . Additionally, SY63 exhibited a higher harvest index than LYPJ under the LN and HN treatments, respectively  and a lower half-filled grain percentage by 10.0 and 7.7%, respectively ; however, such differences were not significant. Higher biomass and grain yield was observed in SY63 than in LYPJ under the LN and HN treatments, but a significant difference was observed only under the LN treatment .SY63 exhibited a significantly higher grain filling percentage and grain weight than LYPJ under both the LN and HN treatments . Soluble sugars and sucrose in stems increased from the date of panicle initiation to 40 days and then decreased until maturity .Stem starch and NSC concentrations in both the SY63 and LYPJ cultivars increased between the panicle initiation and heading stages (approximately 25 days after panicle initiation) under both the LN and HN treatments, and then decreased . Soluble sugars and sucrose were higher through the entire period from panicle initiation to maturity under LN than under HN . The NSC and starch concentrations in SY63 were higher than in LYPJ from panicle initiation to heading and lower after heading under both the LN and HN treatments. From 6 to 24 days after heading, NSC and starch concentrations rapidly decreased in both cultivars and under both N application conditions . Generally, the soluble sugar and sucrose contents of SY63 were higher than in LYPJ from panicle initiation to maturity under the LN and HN conditions .Starch and NSC concentrations were higher from panicle initiation to heading under LN than under HN, and LN accelerated the disappearance of starch and NSC after heading in both LYPJ and SY63  used to describe stem NSC translocation were significantly higher under LN than under HN in both the LYPJ and SY63 cultivars. Additionally, SY63 exhibited higher δCNSC, ATM, AR, and AC than LYPJ, but significant differences were observed only in ATM under both LN and HN applications and in AC under HN application .The four traits .The activities of AGP, StS, and SBE in stems increased from panicle initiation to heading, reaching their peak at approximately 5–10 days before heading and then decreasing. The activities of the three enzymes were higher under LN than under HN in both cultivars and higher in SY63 than in LYPJ under both N applications .The activities of α-amylase and β-amylase in stems increased during grain filling and were enhanced by LN. Additionally, the activity of α-amylase reached a peak 18 days after heading under both the LN and HN treatments, while the activity of β-amylase reached a peak at 24 days after heading. Overall, β-amylase activity was lower than that of α-amylase, exhibiting only small changes during grain filling. The activities of α-amylase and β-amylase were higher in SY63 than in LYPJ under both N applications . However, SSs, AI, NI, and SSc in both cultivars decreased through the grain filling stage under both the LN and HN treatments . Compared with the HN treatment, the LN treatment increased SPS activity in both LYPJ and SY63 ; however, N treatment did not affect activities of SSs, AI, NI, and SSc . In general, SPS activity was higher in SY63 than in LYPJ under both N application treatments . No significant differences in the activities of SSs, AI, NI, and SSc were observed between the two cultivars, except for higher AI activity in LYPJ during the early grain filling stage and higher SSc activity in LYPJ on the day of heading .The activity of SPS first increased, then maintained a relatively high level from 18 to 24 days after heading, and finally declined .The activities of AGP, StS, and SBE were positively correlated with increases in stem starch and NSC during panicle initiation. These correlations were significant for AGP and StS under both N application treatments, but only under the LN treatment for SBE .The activities of α-amylase, β-amylase, and SPS were positively correlated with starch and NSC remobilization after heading. Most of these correlations were significant, but not the correlation between α-amylase activity and starch/NSC remobilization under HN , which was accordant to the previous studies, with the finding that stem NSC accumulation at heading decreased under HN conditions  and varied in consistency with changes in starch and NSC concentrations . Moreover, the activities of AGP, StS, and SBE enzymes positive correlated with starch and NSC concentrations , which agrees with previous results , this result is in accordance with Figures 3A,B), indicating higher starch and NSC remobilization from stems to grains under LN. It has been reported that the remobilization of stem NSC closely correlates with senescence; plants experiencing severe senescence due to environmental stress, such as water stress or LN versus HN, exhibit a greater degree of starch and NSC remobilization than plants experiencing a lower degree of senescence . The increased activities of these enzymes also appeared to enhance remobilization of stem starch and NSC , which was supported by the positive correlation of the activities with the decrease in starch and NSC in stems during grain filling under the LN treatment . Although β-amylase activity was lower than α-amylase activity for the duration of the grain filling stage , β-amylase activity correlated more closely with starch and NSC remobilization . β-amylase is thought to hydrolyze the oligosaccharide products of α-amylase and plays an important role in the regulation of starch hydrolysis ; this result is consistent with previous research showing that SPS is a key enzyme in sucrose synthesis . In contrast, the activity of SSs decreased gradually from heading to maturity and was not affected by N treatment; however, SSs activity was higher than SPS and remained at a high level during grain filling , a state which is beneficial for sucrose synthesis. Overall, N treatment had no effect on the activities of enzymes involved in sucrose hydrolysis  in stems; the activities of these enzymes were relatively low and decreased gradually during grain filling . This suggests that AI, NI, and SSc had little effect on sucrose synthesis and the remobilization of stem starch and NSC. Based on these findings, we conclude that high activities of α-amylase, β-amylase, and SPS are the main contributors to increases in the remobilization of starch and NSC in rice stems under LN during grain filling.Sucrose is the primary form in which photoassimilates are transported in plants . Rapid synthesis . In addiFigure 1). The observation was in accordance with It was noteworthy that superior spikelet had faster grain filling rate and shorter active grain filling period under LN . Higher activities of AGP, StS, and SBE for starch synthesis in stems contributed to the higher stem starch and NSC concentrations in SY63. This result was also supported by the positive correlations between the activities of the three enzymes and the increases in starch and NSC concentrations in the two varieties in present study . Additionally, AGP and StS activities in both cultivars correlated significantly with increases in stem starch and NSC concentrations under both N application treatments ; however, SY63 exhibited strong correlations between SBE activity and increases in starch  and NSC  under both N applications. and no such correlations were observed between SBE activity and increases in starch and NSC for the LYPJ cultivar . Among the starch-synthesis-related enzymes , SBE activity exhibited the highest positive correlation with starch concentration . These results suggest that higher activities of AGP, StS, and SBE may contribute to higher stem starch and NSC concentrations in SY63 than LYPJ and that SBE may contribute more strongly to pre-anthesis starch and NSC accumulation in the stems of SY63 than LYPJ.Rice cultivars have shown large genotypic differences in their abilities to accumulate and translocate stem NSC . The difntration , and SBEntration . Additiontration ; howeverFigures 2A,D,F). Our study and Stem NSC is an important source of assimilates used for grain filling; a high level of stem NSC at the full heading stage was found capable of maintaining enhanced sink strength and highly ripened grains . Grain y-1); however, the biomass and grain yield increased approximately twice between the N treatments , suggesting that the differences in biomass and grain yields between LN and HN were not linearly proportionate with the increase of N application rates. The relationship between N application rate and grain yield in our study was in accordance with lots of previous studies in pot and field experiments. -1) than LN (0.5 g N pot-1) in three rice varieties; Similarly, field experiment data of -1) were 1.7 and 1.5 times higher than those under LN (0 kg N ha-1), respectively. So, the large increases in N application rates do not always result in large differences accordingly in biomass and grain yields. In fact, the responses of rice plant biomass and grain yield to N application rates may be not accumulative, and is associated with several factors, such as soil indigenous nitrogen content (soil fertility), genetic characteristics of cultivars, climate factors (radiation and temperature), cultivation managements.Nitrogen fertilizer application rate has crucial roles in biomass accumulation and grain yield formation. However, increasing fertilization generally result in gradual reduction in the crop yield response . We notiFigure 4), LN enhanced grain filling percentage and harvest index , which may be attributed to the increased stem NSC translocation. Although our study showed that grain yield significantly decreased under LN , investigations by the International Rice Research Institute since 1997 have shown that appropriate reductions in nitrogen fertilizer application can simultaneously increase grain yield and nitrogen use efficiency and reach optimal productivity  and reports of The pursuit of higher rice yields often prompts the application of additional nitrogen fertilizers in many rice planting regions , resultiuctivity . AccordiIn the current study, LN increased the concentrations of starch and NSC in rice stems before the plants’ heading stage. This was primarily attributed to the enhanced activities of AGP, StS, and SBE enzymes in plant stems. Reduced N application also resulted in increased activities of α-amylase, β-amylase, and SPS, which increased the remobilization of stem starch and NSC; these effects increase grain filling percentage, grain filling rate, and harvest index in rice. In terms of cultivar genotype, higher activities of AGP, StS, and SBE were responsible for higher concentrations of stem starch and NSC in the SY63 rice cultivar than in the LYPJ cultivar prior to heading; SBE played a larger role in the regulation of starch and NSC in SY63 than in LYPJ. In addition, SY63 exhibited higher activities of α-amylase, β-amylase, and SPS, which increased stem starch and NSC translocation; these effects increase grain filling percentage, grain filling rate, grain weight, and grain yield in SY63. Our results suggest that calculated reductions in nitrogen application may improve grain yields via enhanced stem NSC accumulation and translocation, thereby reducing the costs and increasing the sustainability of rice production.GL and KC conceived the research, designed the experiments, analyzed the data, and wrote the manuscript. GL carried out pot experiments. QH and YS assisted in both sampling and physiological determinations in the laboratory. SP, JH, and LN gave valuable suggestions during the whole pot and laboratory experiments.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.


Reconstruction of the medial patellofemoral ligament (MPFL) has been increasing as a surgical solution for treatment of recurrent lateral patellofemoral dislocation. Recent attention has been given to fibers extending from the femur to the quadriceps tendon, proximal to the MPFL, termed the medial quadriceps tendon-femoral ligament. This article briefly reviews the proximal medial patellar restraints and surgical procedures for their reconstruction. Knowledge of patellofemoral (PF) joint pathophysiology and kinematics continues to evolve. Several anatomic structures have been identified as playing a role in the biomechanics and pathophysiology of injury to the medial side of this joint. These structures are typically divided into dynamic and static stabilizers. Historically, the static stabilizers have been labeled the patella retinaculum, and include the medial patellofemoral ligament (MPFL) and patellotibial ligament. More recently, the medial patellotibial ligament (MPTL) and medial patellomeniscal ligament (MPML) have gained recognition in literature , 2, as wWith the increasing recognition of the medial patellar restraints, one can divide these ligaments into the proximal medial patellar restraints (MQTFL + MPFL) and the distal medial patellar restraints (MPTL + MPML).Classically, PF joint stabilizing procedures have involved alteration of dynamic stabilizers, in particular the vastus medialis obliquus (VMO) muscle. Literature has shown that static stabilizers, in particular the MPFL, play an important role in patellofemoral biomechanics –7, showiThis article briefly reviews the proximal medial patellar restraints and surgical procedures for their reconstruction.In 1948, Last et al.  describeIn 1979, Warren and Marshall  publisheOver a decade later, in 1993, Conlan et al.  publisheA consistent anatomic feature that can be utilized to locate the MPFL is identification of the “triangle” described by Tuxøe et al.  and discMedial patellofemoral ligament anatomy was further defined by Smirk et al. , who desAn anatomic review of the medial patellofemoral ligament by Amis et al.  noted thNomura et al.  dissecteIn 2007, LaPrade et al.  reportedIn 2009, Baldwin  describeIn the pediatric population, studies consistently cite the MPFL femoral origin distal to the medial distal femoral physis , 29.Recently, attention has returned to the fibers extending from the femur to the quadriceps tendon that have been previously described , 21. TheNear the MQTFL, anatomic descriptions of the MPFL agree on the superior border of the MPFL emanating from the fascial layer on the posterior aspect of the VMO. Therefore, the extent to which the MPFL is “uncovered” in any one specimen depends on the robustness and distal extent of the oblique VMO fibers Fig. . In trauSince its very first description in 1992 by Ellera Gomes et al. , MPFL reThe clinical scenario in which isolated MPFL reconstruction is sufficient to stabilize the patella, without any bony work to compensate or a shallow trochlea, patella alta, or lateralized tibial tubercle, has not yet been categorized with clarity . The oriGraft choiceGraft fixationControlling graft length and its change through the knee arc of motion, which is associated with knee flexion angle at the time of graft fixation and the degree of tautness at the time of graft fixationThe most widely varied aspects of MPFL reconstruction are:Graft choice is an important consideration before MPFL reconstruction. Since the native MPFL has a failure load of approximately 200 N  reconstruction. Moreover, in very specific conditions such as connective tissue disorders , autografts may not provide sufficient tensile strength for effective reconstruction.Current literature shows that graft choice does not impact the outcomes of MPFL reconstruction; the choice is made based upon surgeon preference and experience.Femoral and patellar fixation during MPFL reconstruction represent another matter of debate, since different methods on both sides have been shown to provide good results.Several types of fixation have been described, including use of anchors , 43 or iCurrent clinical and biomechanical studies do not identify the best fixation method on the patellar side. For this reason, the patellar fixation method is based on surgeon preference. High-level comparative studies are needed to determine which type of fixation provides the best functional results and lowest complication rates for various patient groups, patella size, and bone quality.However, the location of MPFL graft fixation on the patella is important. Depending on the fixation technique and patella size, inferior graft placement can create increased distal restraint that could be a cause of reduced knee motion and/or pain in deeper knee flexion. For anatomic reconstruction, the center of the graft fixation should be located at the superomedial aspect of the patella, 27 % from the superior pole (if the distance between the superior and inferior poles is 100 %) . The MPFFemoral fixation methods during MPFL reconstruction can be divided into two basic categories: bone fixation and soft tissue fixation.Bone tissue fixation still represents the most common technique. Considering this type of fixation, the most frequent method is to create a blind bone tunnel at the femoral anatomical insertion, through which the graft is passed and fixed with an interference screw. Reliable alternatives to this kind of fixation are to secure the graft to the femur by using suspensory loop cortical button  or suturThough the first MPFL technique published by Ellera Gomes et al.  utilizedRegardless of the technique used, the femoral location is critical to surgical success. Improper placement of the femoral tunnel may lead to altered patellofemoral biomechanics and, thus, poorer outcomes. The correct location can be found using radiographic or anatomic landmarks Fig. . StephenThe flexion angle during graft fixation helps determine the “tautness” of the graft with knee motion, and is variable in practice, being highly surgeon dependent. The debate centers on whether one believes in MPFL isometry through knee flexion arc with proper femoral fixation. All are in agreement that we need a fixation location and a ligament “tautness” that does not get tight or stretched in flexion, yet maintains an appropriate lateral restraint in early flexion.Different studies have reported several knee flexion angles, including 20° , 30° 4444, 72, 4Tension means the state of being stretched (noun) or applying a force to something that tends to stretch it (verb). Graft tensioning is of paramount importance during graft fixation, and excessive tension should be avoided since it could be associated with stiffness, overcorrection of the patellar tilt, and early progressive degeneration of the medial patellofemoral joint. Though graft tension and the knee flexion angle at time of fixation are related, they are not synonymous. Proper graft tension, and how to achieve this during fixation, remains debatable.In a cadaveric study, Philippot et al.  stated tThe immediate postoperative goal after MPFL reconstruction is to reduce swelling and pain. The goals in the rehabilitative phase are to eliminate pain, regain range of knee motion, and restore quadriceps strength. Postoperative protection of the knee is somewhat dependent on the strength of the graft fixation, but for most current surgical methods, progression is based on functional goals and not time.The training phase involves regaining strength and normal functional gait pattern. For MPFL reconstruction, this is variable but typically completed by 4–6 weeks. The advanced training phase involves examination of complex body movement patterns, primarily to reduce the risk of reinjury and maximize surgical success. This phase is typically 6–12 weeks postoperatively.Regarding return to sport, the injured patient is often eager for an expected timeline for return to full function. Estimates can be given, but the athlete and physician must be aware that the ultimate return to activities, be they daily activities or higher-level competition, depends on recovery of strength, flexibility, and appropriate body movement patterns.Various functional tests, more commonly used in knee ligament surgery, can be employed to aid estimation of patient readiness to return to activities. The average return to sport, depending on patient- and sport-specific factors, is 4–6 months.An explosion of knowledge based on PF anatomy and biomechanics principles has positively influenced treatment of PF injuries, in particular lateral patellar dislocation. Patella stabilization surgery demands broad knowledge of anatomic and biomechanical principles as reviewed herein.


Acinetobacter species and Pseudomonas species, especially multidrug-resistant (MDR) strains pose a serious management challenge with a public health threat.Infections caused by Acinetobacter spp or Pseudomonas aeruginosa was carried out at Manipal Teaching Hospital from 2014 to 2016.A hospital-based retrospective study of patients who were infected with Acinetobacter spp. and 313 cases with Pseudomonas aeruginosa were studied. The rate of nosocomial infections was higher than non-nosocomial infections. ICU was found as the major hub for both the organisms; (53.5% of cases due to Acinetobacter spp. and 39.6% due to Pseudomonas aeruginosa). Most isolates were of respiratory tract origin (Acinetobacter 74.7% and Pseudomonas aeruginosa 65.8%). Percentage resistance of Acinetobacter spp. towards polymyxin B was found to be quite low (18.8%). Similarly, resistance rates of Pseudomonas aeruginosa against amikacin were also found to be low, i.e., 17.4%. A higher prevalence of multidrug resistance was seen among Acinetobacter spp than among Pseudomonas aeruginosa (75.9% vs. 60.1%). The hospital stay was longer for patients infected with MDR isolate (p=0.001 for Acinetobacter spp. and p=0.003 for Pseudomonas aeruginosa). The mortality rate was higher in infections due to Acinetobacter spp (15.9%) as compared to Pseudomonas aeruginosa (8.3%).A total of 170 cases of infections with Acinetobacter species and Pseudomonas aeruginosa are associated with prolonged hospital stay and high in-hospital mortality. These emphasize the need for prudent use of antibiotics and aggressive infection control strategies.This study reveals that infections caused by  Acinetobacter and Pseudomonas species are gram-negative bacilli that commonly cause healthcare-associated infection. These can survive for prolonged periods in the environment and the hands of healthcare workers  and Pseudomonas aeruginosa for 36.1% [113 of 313] of the cases . Amongst all Pseudomonas aeruginosa isolates, however, a significant proportion, i.e., 285 (91.2%) were resistant to cefixime, whereas very few  showed resistance to amikacin. Antibiotic susceptibility pattern is shown in It is noteworthy to mention that quite a high proportion (75.9%) of Acinetobacter spp. than with P. aeruginosa (15.9% vs. 8.3 %) [p value=0.085 for Acinetobacter spp. & 0.064 for P. aeruginosa). The mortality rate was two-fold higher among those infected with MDR organisms  were longer among patients yielding MDR bacteria than those yielding non-MDR bacteria on culture. Mortality was higher among patients infected with rganisms  when comPseudomonas aeruginosa ranked second among the gram-negative pathogens reported to the National Nosocomial Infections Surveillance System (NNIS) [Acinetobacter spp were on an increasing trend each year. Acinetobacter species caused 7% of ICU healthcare-associated pneumonia in 2003 compared with 4% in 1986, as reported by the National Nosocomial Infections Surveillance System [Acinetobacter spp. has been associated with infections in critical care patients, especially with ventilator-associated pneumonia. In complying with other studies, we also found a high rate (78.2%) of nosocomial infections was due to Acinetobacter spp. [Acinetobacter spp. This is in contrast to the findings of Mishra et al. [Acinetobacter species. Such a high rate of detection of MDR isolates in their study was attributed to Extended-spectrum β-lactamase (ESBL) and AmpC beta-lactamase production [Acinetobacter spp. between our study and theirs’ could be due to the differences in adapting the antibiotic policies over the period.m (NNIS)  in causie System . Acinetoter spp. . As repoa et al. , who notoduction . The disAcinetobacter species worldwide. We found ciprofloxacin resistance in 67.6% of the isolates. This is in agreement with the results of Joshi et al. from India [Acinetobacter isolates. By the observations noted above [Acinetobacter amongst the inpatients is always challenging before the clinician. In this context, other options, such as beta-lactam and beta-lactamase inhibitor combinations, could be the alternative therapeutic regime. We explored the scenario of the susceptibility pattern of the Acinetobacter spp. towards beta-lactam beta-lactamase inhibitor combinations. It was revealed that 48.4% of the Acinetobacter spp. were resistant to cefoperazone-sulbactam and 63% to piperacillin-tazobactam. Ling et al. [Acinetobacter were susceptible to cefoperazone-sulbactam combination than to piperacillin-tazobactam (91% vs. 21%).Presently, fluoroquinolone resistance is being recognized as an emerging problem in om India , and Misom India  who repoed above , 13-16, g et al.  from ShaAcinetobacter spp. towards carbapenem (38.2%) was comparable to that reported from India (34%), although a recent study from Nepal documented almost 50% of the clinical isolates to be resistant to this drug [Resistance rate of his drug . Howeverhis drug . ConsideAcinetobacter isolates were susceptible to Polymyxin B. Though this drug is often, not preferred in clinical practice due to its nephrotoxicity and neurotoxicity [A. baumannii [It was encouraging to note that a maximum number  of toxicity  yet thertoxicity . Neverthtoxicity . Tigecycaumannii . But botAcinetobacter infections had increased length of hospital stay as compared to those infected with non-MDR Acinetobacter (19 days vs. 13 days). This finding combined with increased risk for in-hospital transmission of the organism [Acinetobacter spp. in health care settings. In the present study, as high as 15.9% mortality rate was noted among the cases infected with MDR Acinetobacter. Sunenshine et al. [Acinetobacter.We found that patients having MDR organism  supportsPseudomonas aeruginosa isolates were from the Medical ICU (39.6%). Our result was in consistence with the previous studies [Pseudomonas spp. with a direct impact on mortality. Infections caused by Pseudomonas aeruginosa is frequently life-threatening and often difficult to treat because of the intrinsic susceptibility of Pseudomonas aeruginosa only to a limited number of antimicrobial agents [P. aeruginosa isolates to be MDR [Pseudomonas aeruginosa were MDR. This discrepancy could be because the organisms tested in the Kathmandu study [Most of the  studies , 19. Mull agents , 21. A so be MDR . We, howdu study  were froPseudomonas aeruginosa were susceptible to amikacin and piperacillin-tazobactam respectively, only 8.8% had shown susceptibility towards cefixime. Bhandari et al. [Pseudomonas aeruginosa as susceptible to cefoperazone-sulbactam, 54.5% to piperacillin-tazobactam, and 51.5% to meropenem. They found a maximum number of the organisms showing resistance to cefixime (93.9%), a finding very much similar to ours . The length of hospital stay was higher for patients having infections due to MDR Pseudomonas aeruginosa than due to non-MDR Pseudomonas aeruginosa (21 days vs. 13 days). Previous workers also reported similar findings [Pseudomonas aeruginosa was shown to be 21% in another study conducted by Gyanes et al. [Whereas 82.6% and 80% of i et al.  from Nepfindings . In our s et al. .Acinetobacter species and Pseudomonas aeruginosa are associated with prolonged hospital stay and high in-hospital mortality. These emphasize the need for prudent use of antibiotics and aggressive infection control strategies.This study reveals that infections caused by Acinetobacter and Pseudomonas aeruginosa infections among ICU patients in our setting.Our study provided adequate information on the high prevalence of MDR, as well as pan drug-resistant Ample informative data on patients’ clinical details were not available to see any correlation between multidrug resistance and type of the organism, with the severity of illnessSusceptibility towards antibiotics like tigecycline and colistin were not tested.Antibiotic resistance, including multidrug resistance, is the leading cause of mortality and morbidity in hospitalized patients. Further initiatives are needed to run continuous surveillance programs to monitor drug resistance patterns among these isolates to formulate measures for effective control of antibiotic resistance.Acinetobacter and Pseudomonas are available from different parts of Nepal.Few studies on antibiotic resistance patterns of Acinetobacter spp. and Pseudomonas aeruginosa isolated from hospitalized patients.The study adds the information on the current antibiogram profile of


Grain legumes represent a valuable energy source in pig diets due to their high starch content. The present study was conducted to determine the content and apparent ileal digestibility (AID) of starch in different grain legume cultivars for pigs by means of both a polarimetric and enzymatic method for starch determination.Vicia faba L.) and peas (Pisum sativum L.), five different cultivars of lupins , and one diet with a soybean meal (SBM) were fed.Three experiments were conducted with six barrows each which were fitted with ileal T-cannulas. In total, 18 diets including six different cultivars of faba beans (P < 0.05) when determined polarimetrically than enzymatically (438 vs. 345 g/kg dry matter (DM) in faba beans and 509 vs. 390 g/kg DM in peas, respectively). Considerable lower starch contents were obtained in lupins and SBM, with 82 and 48 g/kg DM  and <1.1 and 3 g/kg DM , respectively. Mean values for contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in grain legumes ranged from 111 and 79 g/kg DM in peas to 248 and 207 g/kg DM in lupins, respectively. Contents of condensed tannins in the colored flowered faba bean cultivars ranged from 2.1 to 7.4 g/kg DM. The AID of starch was greater (P < 0.05) in pea than in faba bean cultivars, and using the polarimetric starch determination method resulted in greater (P < 0.05) digestibility values than using enzymatic starch analysis (84 vs. 80% in faba beans and 86 vs. 83% in peas). Moreover, AID of starch differed (P < 0.05) within pea cultivars and starch digestibility in faba beans decreased linearly (P < 0.05) as the content of condensed tannins increased. However, there was no relationship between contents of NDF and ADF and AID of starch in pea and faba bean cultivars.The starch content of faba beans and peas was greater (Both contents and AID of starch in grain legumes can vary as influenced by the analytical method used for starch determination. Generally, starch digestibility is greater when measured by polarimetric rather than enzymatic methods. The search for alternative protein sources in livestock nutrition has resulted in growing interest in the use of grain legumes as alternatives for animal by-products or oilseed products such as soybean meal (SBM). In addition, grain legumes have proven to be a valuable energy source in animal nutrition due to their high content of starch in faba beans and peas and lipids in lupins . Accordiα-amylase has been reported to be inversely related to the amylose content, with high amylose starches being particularly resistant [In general, legume starch contains 30 to 40% amylose and 60 to 70% amylopectin, whereas cereal starch consists of 20 to 25% amylose and 75 to 80% amylopectin [As starches in grain legumes are closely associated with proteins, the determination of starch in grain legumes may be confounded by the analytical method used , mainly Vicia faba L.) and peas (Pisum sativum L.), five seed-grade cultivars of lupins (Lupinus L. spp.), and one commercially available SBM were used in three consecutive experiments with six growing barrows (German Landrace × Piétrain) each. The experiment was arranged as a row-column design. Each of the three experiments included six pigs (rows) and six periods (columns). Within each of the three experiments, the animals were randomly allocated to the 18 assay feed ingredients in periods one to three and four to six, respectively, resulting in two replications per experiment and a total of six observations per assay feed ingredient throughout all three experiments. The average initial and final body weight (BW) of the pigs at the beginning and end of the experiment was 23 ± 2 kg and 45 ± 4 kg, respectively. The pigs were surgically fitted with T-cannulas at the distal ileum according to the procedures described by Li et al. [A total of 18 feed ingredients including six seed-grade cultivars of faba beans , acid detergent fiber (ADF), condensed tannins, and titanium dioxide were determined as described by Jezierny et al. .The starch contents in the feed ingredients, the diets, and in ileal digesta samples were analyzed both polarimetrically and enzymatically. The polarimetric method represents the official starch determination method in the European Union for feed according to the Commission Directive 1999/79/EC , and comAi = apparent ileal starch digestibility in the ith diet (%), IAi = marker concentration in the ith diet (g/kg DM), SDi = starch content in digesta of the ith diet (g/kg DM); SAi = starch content in the ith diet (g/kg DM), and IDi = marker concentration in digesta of the ith diet (g/kg DM).The AID of starch in the diets was calculated using the following equation:I = apparent ileal starch digestibility in the feed ingredient (%), IDAi = apparent ileal starch digestibility in the diet (%), IDC = apparent ileal digestibility of corn starch (%), CC = contribution level of starch from corn starch to the diet (%) and CI = contribution level of starch from the feed ingredient to the diet (%).The AID of starch in the feed ingredients was calculated according to the following equation:t-test with degrees of freedom determined by the Kenward-Roger method [F-test [Homogeneity of variances and normal distribution of the data were confirmed and experimental data were analyzed using the MIXED procedure of SAS . The linr method . This te [F-test  revealed [F-test . The sigAnalyzed contents of starch, both determined polarimetrically and enzymatically, and contents of NDF and ADF in grain legume cultivars as well as condensed tannins in faba bean cultivars are presented in Table Within pea and faba bean cultivars, starch contents ranged between 495 and 535 g/kg DM for cultivars Rocket and Hardy, respectively, and between 421 and 456 g/kg DM for cultivars Fuego and Aurelia, respectively, when determined polarimetrically. Using the enzymatic approach, starch contents within pea cultivars ranged from 372 to 423 g/kg DM for cultivars Santana and Hardy, and within faba bean cultivars from 311 to 393 g/kg DM for cultivars Espresso and Limbo. Within lupin cultivars, starch content ranged from 56 in Bornal to 96 g/kg DM in Boregine when determined polarimetrically. However, starch content for all lupin cultivars was below the detection limit of 1.1 g/kg DM when analyzed enzymatically. The starch content in SBM amounted to 48.0 and 3.0 g/kg DM, based on polarimetric and enzymatic measurements, respectively. Contents of NDF and ADF in faba bean cultivars ranged from 126 to 165 and 101 to 137 g/kg DM, respectively, and in the pea cultivars from 104 to 126 and 68 to 86 g/kg DM, respectively. Condensed tannins contents in faba beans amounted to 2.1, 4.2, 7.0, and 7.4 g/kg DM in Divine, Espresso, Limbo, and Fuego, respectively. In peas and lupins, condensed tannins were not detected.P < 0.05). Average starch contents in pea, faba bean, and lupin cultivars amounted to 509, 438, and 82 g/kg DM when analyzed polarimetrically and 390, 345, and <1.1 g/kg DM, respectively, when analyzed enzymatically. The mean polarimetric analyzed starch values in grain legumes were greater when compared to the mean starch contents that were analyzed enzymatically (P < 0.001)  for cultivar Harnas (89%) in comparison to cultivars Phönix (83%), Rocket (83%), and Hardy (85%), when the polarimetric method was used for starch analysis. Based on the enzymatic procedure, AID of starch within peas was greater (P < 0.05) for cultivar Jutta (88%) when compared to cultivars Hardy (82%), Phönix (80%), and Rocket (77%). Apparent ileal starch digestibility in faba beans did not differ (P > 0.05) between cultivars, and ranged from 81 (Limbo) to 86%  using the polarimetric starch determination method and from 75 (Limbo) to 82%  with the enzymatic starch determination procedure.Within pea cultivars, AID of starch was greater (P < 0.05) in faba beans when compared to pea cultivars (80 vs. 83%), if starch contents were analyzed enzymatically. Similarly, mean values for AID of starch of faba beans and peas were lower (P < 0.05) when starch content was determined enzymatically rather than polarimetrically.Mean values for AID of starch did not differ between faba bean and pea cultivars (84 vs. 86%) when the polarimetric method was used Table . Howeverß-glucans are hydrolyzed [Mean values for polarimetrically analyzed starch contents of peas were within the range of previously reported data , 23. Basdrolyzed . Similardrolyzed  reporteddrolyzed .Differences in starch contents within grain legume cultivars may be due to differences in genetics, but also to varying growing and harvesting conditions , 32. JørIn the present study, AID of starch of faba beans was up to 13 percentage units lower compared to AID values obtained by Jansman et al.  in pigleP < 0.05), but there are no differences over the entire digestive tract [Hydrolysis of starch in the gastrointestinal tract is influenced by a number of host factors, in addition to differences in physical characteristics of the ingested food and starch. Host factors, for example, include intensity of chewing the feed, the availability of pancreatic α-amylases, and digesta retention time in the small intestine . Starch ve tract . Consequve tract , up to 2Differences in AID of starch have been attributed to the existence of an enzyme-resistant starch fraction, differences in starch digestion rate, or both . It is gGrain legume starches are generally part of the protein matrix . Most ofPhaseolus vulgaris), amylolysis was enhanced by wet homogenization and pepsin pretreatment [Differences in AID of starch between pea cultivars in the present study may also be attributed to the presence of cell wall components surrounding the starch granules, thereby inhibiting access of amylases to the granule. According to results of in vitro digestion of starch in red kidney beans (reatment . These fP < 0.05) with increasing tannin content of the diets . Differences between AID of starch in faba beans and peas or within pea cultivars may be attributed to variations in contents of resistant starch. Moreover, variations in amylose to amylopectin ratio of grain legumes may, at least in part, explain the observed differences in AID of starch.In faba beans, condensed tannins reduce AID of starch in pigs (e.g. ). CondenStarch contents and AID of starch in grain legumes obtained by means of the polarimetric method are greater than starch values determined enzymatically. This methodical difference is apparently due to the presence of other feed components such as carbohydrates that interfere with the polarimetric starch analysis and (or) has to be attributed to incomplete starch hydrolysis when the enzymatic procedure is applied. Differences in AID of starch between faba beans and peas are confined to the use of the enzymatic method for starch determination. Within pea cultivars, differences in AID of starch were observed for both starch analysis methods. There was a negative linear effect of condensed tannins in the diet on AID of starch in faba beans when the polarimetric method was used for starch determination. The polarimetric approach is the official method used in the European Union for starch determination in feed. However, there is increasing evidence that the enzymatic method is superior over the polarimetric method due to its enzyme specificity for starch.


BackgroundThe Accreditation Council for Graduate Medical Education and the American Osteopathic Association recently agreed to establish a single graduate medical education system for the United States allopathic and osteopathic resident physicians by 2020. Consequential to this merger, new standards will be implemented for academic and research requirements within medical schools as well as residency programs. In the United States, osteopathic medicine is considered to be a parallel profession to allopathic medicine. However, recent studies have revealed that the percentages of United States osteopathic physicians currently in practice are not proportional to the percentages of editorial board member positions they hold in several high-profile medical journals as well as neurosurgical journals. To our knowledge, there is currently no published literature examining osteopathic physician author representation of any neurosurgical journal. In the present study, we analyze the number of osteopathic physicians and osteopathic neurosurgeons serving as authors in prominent neurosurgical journals.MethodsJournal of Neurosurgery, Journal of Neurosurgery: Spine, Journal of Neurosurgery: Pediatrics, and Neurosurgical Focus) fulfilled these criteria. The number of allopathic and osteopathic physicians who have published at least one manuscript in a Journal of Neurosurgery Publishing Group journal was counted. The specialty of each osteopathic author was examined.American neurosurgical journals with the highest number of citations plus an affiliation with a neurosurgical society open to osteopathic neurosurgeons were used as criteria for journal selection. The Journal of Neurosurgery Publishing Group journals  and osteopathic physicians represented 335 (0.32%) of the 105,492 authorship positions held by these physicians in these journals since 1944. Statistical significance was found comparing the number of allopathic authors versus the number of osteopathic authors (p < 0.0001). The most common specialty represented by osteopathic authors in all journals was neurosurgery (45%). Osteopathic neurosurgeons represented 153 (0.15%) of the total number of allopathic and osteopathic authors.ConclusionsThese data establish that the percentages of the United States osteopathic physicians and osteopathic neurosurgeons currently in practice are not proportional to the percentages of authorship positions they hold in Journal of Neurosurgery Publishing Group journals. We postulate that this apparent disproportionality may originate from significant differences between allopathic and osteopathic medical school research funding, research opportunities, scholarly activities, and dual-degree programs. Osteopathic medicine was founded more than 125 years ago in Kirksville, Missouri. Since then, the number of practicing osteopathic physicians in the United States (US) has increased considerably. In 2016, 67,534 (7.7%) of the active US physician population were osteopathic physicians, 586,287 (66.8%) were US-trained allopathic physicians, and 214,681 (24.5%) were international medical graduates [Journal of Neurosurgery fulfilled both criteria. However, we included all Journal of Neurosurgery Publishing Group (JNPG) journals  to broaden our scope of analysis to four journals. Beginning with the inaugural issue of each journal, allopathic and osteopathic physicians who have published at least one manuscript in a JNPG journal were counted. This analysis included each monthly issue in Journal of Neurosurgery from 1944 to May 2018, Neurosurgical Focus from 1996 to May 2018, Journal of Neurosurgery: Spine from 1999 to May 2018, and Journal of Neurosurgery: Pediatrics from 2004 to May 2018. Authors’ positions on manuscripts  were not specified. An Internet search was performed to determine the medical specialty of each author holding an osteopathic degree. The specialties of osteopathic authors were analyzed. Descriptive statistics were utilized on the collected data to determine the number of allopathic and osteopathic physicians serving as authors in each of the four journals. A paired t test was then performed to compare the number of allopathic authors versus the number of osteopathic authors.American neurosurgical journals with the highest number of citations as per Thomson Reuters’ Journal Citations Reports plus an affiliation with a neurosurgical society open to osteopathic neurosurgeons were used as criteria for journal selection. The Journal of Neurosurgery, Journal of Neurosurgery: Spine, Journal of Neurosurgery: Pediatrics, or Neurosurgical Focus). Allopathic physicians represented 105,157 (99.68%) and osteopathic physicians represented 335 (0.32%) of the 105,492 authorship positions held by these physicians. Of the 105,492 positions, osteopathic physicians accounted for 89 (0.14%) of the total number of authors in Journal of Neurosurgery, 86 (0.53%) of the authors in Journal of Neurosurgery: Spine, 105 (0.87%) of the authors in Journal of Neurosurgery: Pediatrics, and 55 (0.45%) of the authors in Neurosurgical Focus . Since the inception of each journal, we found that representation of osteopathic physician authorship has steadily increased , five osteopathic physicians served as authors to Neurosurgical Focus compared to 17 from 2014 to 2018. From 1999 to 2003 , zero osteopathic physicians served as authors to Journal of Neurosurgery: Spine compared to 47 from 2014 to 2018. From 2004 to 2008 , 32 osteopathic physicians served as authors to Journal of Neurosurgery: Pediatrics compared to 43 from 2014 to 2018.The first osteopathic physician to serve as an author in a JNPG journal was in 1978 in Journal of Neurosurgery, 32 (0.20%) of authors in Journal of Neurosurgery: Spine, 68 (0.57%) of authors in Journal of Neurosurgery: Pediatrics, and 27 (0.22%) of authors in Neurosurgical Focus. Collectively, the top specialties of osteopathic authors represented in the journals included neurosurgery, neurology, orthopedic surgery, diagnostic radiology, radiation oncology, and physical medicine . Comparatively, osteopathic neurosurgeons constituted 29% of osteopathic authors in Journal of Neurosurgery, 37% in Journal of Neurosurgery: Spine, and 49% in Neurosurgical Focus.We then sought to examine the medical specialty of each osteopathic physician serving as author in each journal. The most common specialty represented by osteopathic authors was neurosurgery. Osteopathic neurosurgeons comprised 26 (0.04%) of the combined osteopathic and allopathic authors in To our knowledge, the present study is the first to analyze osteopathic physician author representation of any neurosurgical journal. Although osteopathic physicians constitute 7.7% of the active US physician population, we report that they only comprise 335 (0.32%) of 105,492 authorship positions held by allopathic and osteopathic physicians in JNPG journals [The underrepresentation of osteopathic physician and osteopathic neurosurgeon authorship in these journals may be attributed to a multitude of factors beginning with significant differences between allopathic and osteopathic medical school research funding, scholarly activity, and dual-degree programs. Compared to their allopathic counterparts, osteopathic medical school funding from the National Institute of Health (NIH) is insignificant. Indeed, allopathic medical schools received 800 times more funding from the NIH than osteopathic medical schools in 2011 . FurtherWith inadequate research opportunities for medical students to take part in, student’s residency applications may become deficient in scholarly activities – a key factor for resident selection. From 2006 to 2010, 28 osteopathic medical schools collectively published 1843 manuscripts: approximately 13 publications per year per school . StudentMedical school NIH funding translates into medical student research opportunities, scholarly activities, and the beginnings of a career in academic medicine. The absence of a thriving research environment encountered in osteopathic medical schools may impede the development of academic osteopathic physicians and, therefore, constrain osteopathic physicians from publishing during their career. Collectively, these data not only signify that nearly half of osteopathic medical students are not content with the research experiences (or their lack of) offered by osteopathic medical schools but also provide a foundational basis for the lack of proportionality between the percentages of practicing osteopathic physicians and osteopathic neurosurgeons and authorship positions they hold in JNPG journals.Neurosurgery was the most common specialty represented by osteopathic authors. As such, an examination of research endeavors held by allopathic and osteopathic neurosurgery residency applicants is warranted. The most recent data on charting outcomes for both ACGME and AOA residency applicants was published in 2014. According to the National Resident Matching Program (NRMP) 2014 Charting Outcomes, the mean number of research experiences and scholarly activities  for US allopathic seniors matching into ACGME neurosurgery programs were 4.4 and 11.7, respectively . The 201A major limitation of the current study is that only four neurosurgery journals were examined. The number of osteopathic physician authors may have been higher if additional journals were surveyed. Ideally, the percentage of osteopathic authors should be calculated with the number of American allopathic and osteopathic authors in the denominator. However, it was not feasible to look up the country of origin of 105,157 allopathic authors. All osteopathic physician authors are US-based but not all allopathic physician authors are. Thus, the total number of allopathic authors was counted without specifying American versus international origin. This may have made a slight difference in the percentage of osteopathic author representation in the journals. Nevertheless, JNSPG journals are American-based journals and the majority of authors’ country of origin is the US. Still, the validity of our results remains unchanged even if 90% of allopathic authors were found to be international allopathic physicians.In the present study, we observed a lack of proportionality between the percentages of practicing osteopathic physicians and osteopathic neurosurgeons and authorship positions they hold in JNPG journals. We provided several explanations for the disparity observed including remarkable differences between allopathic and osteopathic medical school NIH funding, research opportunities, and scholarly activities. The absence of a thriving research environment encountered in osteopathic medical schools may impede the development of academic physicians and, therefore, constrain osteopathic physicians from publishing during their careers. These data demonstrate that osteopathic medical schools are trailing far behind their allopathic counterparts in terms of research endeavors and manuscript authorship. With the ACGME-AOA merger in progress and residency spots becoming more competitive each year, osteopathic medical schools ought to correct this deficit for the sake of their graduating students and for the sake of their profession.


The present study sought to introduce clinical characteristics and stepwise surgical strategies of isolated complete cryptophthalmos, a rare, congenital ocular anomaly.Retrospective, noncomparative, clinical study. Six patients with isolated complete cryptophthalmos were diagnosed at the Beijing Tongren Hospital between 2010 to 2018. The presentation age of patients ranged from 1 month to 68 years. This study includes two males and four females, and involvement was noted to be bilateral in two cases and unilateral in four cases. According to orbital CT scan and ocular CDI results, two patients were combined with ocular cyst. Reconstruction surgeries were performed in three patients, involving the eyeball enucleation, creation of fornix, eyelid reconstruction with skin flaps/amniotic membrane, and implantation of prosthesis. Besides, implantation of hydroxyapatite was performed in one pediatric patient to promote orbit development. Good outcomes in terms of cosmetic satisfaction were achieved in all patients during follow-up.Surgical intervention could only improve the cosmetic appearance in isolated complete cryptophthalmos. The surgical strategies may be planned to use three-stage approaches described in this study. Meanwhile, orbital development must be taken into consideration in pediatric cases. Cryptophthalmos, first described by Zehender in 1872, is an extremely rare, autosomal recessive ocular disorder and may occur in isolation or as a part of Fraser syndrome.  In complRehabilitation of cryptophthalmos presents an extremely challenging task for ophthalmologists.  In most The medical records of patients with isolated complete cryptophthalmos who were referred to Beijing Tongren Hospital between 2010 and 2018 were reviewed. All patients were examined and managed by the same oculoplastic surgeon. Data were documented including the age at presentation, gender, consanguinity, family history, clinical presentations, surgical interventions, and outcomes. Patients’ images were taken for confirmation of the physical findings. In addition to ophthalmologic examination, patients received ocular CDI and orbital CT scan preoperatively. The study adheres to the tenets of the Declaration of Helsinki for research involving human subjects and was approved by the ethics committee of Beijing Tongren Hospital. Written informed consent from patients or their parents was obtained for publication of potentially identifying details as well as the images in this case series.As can be seen in Table A four-month-old girl born with absent eyelids, anterior hairline anomaly on the left side, and her eyebrow was replaced by a downward sweep of the frontal hairline and a mass which was gradually increasing in size from the left orbit. Fig. a The rigThe first stage of surgical interventions included enucleation of cystic eye and hydroxyapatite (HA) implantation were performed. Fig. c-e FolloAn eleven-month-old girl was born with bilateral absent eyelid, aberrant hairline and eyebrow anomaly. Fig. a During A twenty-four-year-old male patient was presented with absent eyelids and aberrant eyebrow and a horizontal skin scar can be seen in Fig. Cryptophthalmos, which means “hidden eye” in Greek, was first introduced by Zehender and he described a pediatric patient whose eyes were totally covered by skin.  And ManzIsolated cryptophthalmos must be differentiated from Fraser syndrome. Fraser syndrome, a rare, autosomal recessive systemic disease, was first described by Canadian geneticist Fraser in 1962. , 8 as ouComplete cryptophthalmos is believed as an extremely rare, autosomal recessive ocular disorder. In 1986, Thomas reported a review of 124 cryptophthalmos cases which include 27 isolated cryptophthalmos and 97 syndromic cryptophthalmos. , 9 One sThe exact causes of complete cryptophthalmos remain uncertain. From embryological perspective, cryptophthalmos can be explained due to failure in the movement of embryologic surface ectoderm and mesodermal nonpenetration.  EmbryoloRehabilitation of cryptophthalmos is a challenging scenario with no agreed guidelines for its management. , 2, 4 GeIn the second step, the incision is reopened to construct the eyelid margins 1 yr after the first step of surgery. And the lamellae of the eyelids are preferably stiffened with an ear cartilage  or bankeIn the third step, retain-wearing artificial eyes were employed to improve cosmetic effect. Patients were followed up at regular intervals with the assessment of the post-operative cosmetic and functional outcomes. Meanwhile, the orbital development in pediatric patient should be highly-considered. During the follow-up, retrobulbar injection of hydrogel can be adopted to orbital expansion  in pediaIn summary, oculoplastic rehabilitation in isolated complete cryptophthalmos presents specific challenges and the primary objective of surgical intervention is to improve the cosmetic appearance. The reconstruction frequently entails stepwise approaches including enucleation, reconstruction of eyelids, and further implantation of prosthesis. Additionally, orbital development should be taken into consideration in pediatric cases. To our knowledge, this is one of the largest studies to reveal several key properties of isolated complete cryptophthalmos, including clinical features, surgical interventions and long-term cosmetic outcomes.