Abies faxoniana is the dominant plant species of the forest ecosystem on the eastern edge of Qinghai-Tibet Plateau, where the treeline is strongly defined by climate. The tree-ring chronologies and age structure of Abies faxoniana were developed in the treeline ecotones on the northwestern and southeastern aspects of the Min Mountains in the Wanglang Nature Reserve to examine the treeline dynamics of recent decades in response to climate change.On the northwestern aspect, correlation analysis showed that the radial growth was significantly and positively correlated with precipitation in current January and monthly mean temperature in current April, but significantly and negatively correlated with monthly mean temperature in previous August. On the southeastern aspect, the radial growth was significantly negatively correlated with monthly mean temperature in previous July and August.The different responses of radial growth to climatic variability on both the aspects might be mainly due to the micro-environmental conditions. The recruitment benefited from the warm temperature in current April, July and September on the northwestern aspect. The responses of radial growth and recruitment to climatic variability were similar on the northwestern slope. Recruitment was greatly restricted by competition with dense bamboos on the southeastern aspect.The online version of this article (doi:10.1186/1999-3110-54-15) contains supplementary material, which is available to authorized users. Upper treeline ecotones within alpine and arctic ecosystems are mainly controlled by climate  in the Min Mountains, on the eastern edge of QTP in western Sichuan Province, southwestern China   were established at the subalpine treeline ecotones on the northwestern  and southeastern  aspects of the Min Mountains in the Wanglang Nature Reserve  were obtained from the nearest meteorological station, Songpan , approximately 55 km southwest of the sampling sites. Monthly mean temperature and monthly total precipitation of the sampling sites were simulated by Mountain Climate Simulator  based on daily maximum and minimum temperature and precipitation of base station  and height  of the fir seedlings/saplings  on both northwestern and southeastern aspects are shown in Figure The age structures of On the southeastern aspect, the 1-10-year-old age class (fir trees that established in the 2000s) formed the largest age class (11%). The majority of fir trees (78%) were successfully recruited into the treeline ecotone from 1750 s to 1900s Figure b. HoweveThe short-time recruitment records in recent decades did not match with the available meteorological records (1951–2009) in the treeline on the southeastern aspect Figure b, so we During the last 60 years, significantly positive correlations existed between recruitment and monthly mean temperatures for the current April, July and September. However, monthly precipitation had no significant correlations with the recruitment Figure .Figure 7A. faxoniana was positively correlated with the temperature in current March at the treeline site in Wolong National Natural Reserve , which lies west of our study area. Warmer temperatures early in the growing season benefit A. faxoniana growth by inducing early snowmelt and increasing available soil water (Wang et al., Generally, the responses of radial growth to temperatures in previous summer were similar at the treeline ecotones on both the aspects. Significant negative correlations with previous July/August mean temperatures likely reflected the negative carry-over effects Figure . NegativA. faxoniana might reduce solar radiation and effective photosynthesis, thereby, shorten the growing season (Wang et al., The correlations between the precipitation and radial growth were different on both aspects Figure . There wThe differential responses of radial growth to climate factors on both sites might be mainly due to the difference in micro-environmental conditions between the contrasting slope aspects. The studied area is dominated by the southeast monsoon (Pu et al., A. faxoniana at the treeline of the Min Mountains on the eastern edge of QTP might be controlled mainly by temperature, wind, snowpack depth and winter drought.The age structure of a stand can provide a fairly accurate picture of temporal variations in the recruitment rate Kullman,  with theAbies spectabilis forest in the alpine timberline of the Mt. Everest in southern QTP, China (Lv and Zhang, Recruitment was mainly affected by the temperatures in the spring, summer and early autumn seasons, and it was found to have significantly positive correlations with mean temperatures in April, July and September Figure . TemperaThere was a large gap in recruitment during the middle of the 20th century on the southeastern aspect Figure . CompetiA. faxoniana had no obvious increasing trends with small fluctuations on both the aspects (Figure A. spectabilis on the southern QTP (Lv and Zhang, Picea schrenkiana and Abies fargesii in response to warm climate over several of the most recent decades. The different results suggest that the response of treeline ecotones to climate change varies with both local site conditions and the individual species (Luckman and Kavanagh, Over the most recent decades, the radial growth of s Figure . Howevers Figure a. Obvious Figure , Swedishs Figure  and U.S.s Figure . Our resA. faxoniana seedling recruitment were also similar to those that enhanced the radial growth of fir trees. For example, high temperatures in current April enhanced the recruitment of A. faxoniana and facilitated the radial growth (Figures At many upper treeline ecotones the climatic conditions that facilitate seedling recruitment are frequently similar to those conducive to radial growth of trees (Szeicz and Macdonald,  Figures  and 7; m Figures  and 7.A. faxoniana in the treeline ecotones on the eastern edge of QTP. For the geographical novelty, QTP is attracting more and more attention on conducting similar studies (Liang et al., In summary, we investigated the climatic response of radial growth and recruitment of


The expression of CD40, CD80, CD83, and CD86 were upregulated in peripheral blood from the DC + TL + TRF group compared to other groups. In addition, there was higher expression of FasL in tumor-excised mice from the DC + TL + TRF group compared to other groups. FasL plays an important role in maintaining immune privilege and is required for cytotoxic T-lymphocyte (CTL) activity. Microarray analysis identified several genes involved in the regulation of cancer. In this study, we focused on the special AT rich binding protein 1 (SATB1) gene, which was reported to have dual functions, one of which was to induce aggressive growth in breast cancer cells. Tumors from DC + TL + TRF mice showed lower (p < 0.05) expression of SATB1 gene. Further study will be conducted to investigate the molecular functions of and the role of SATB1 in 4T1 mammary cancer cells and DC. In conclusion, TRF supplementation can potentiate the effectiveness of DC-vaccine immunotherapy.The aim of this study was to evaluate the effectiveness of immunotherapy using dendritic cells (DC) pulsed with tumor lysate (a DC vaccine) in combination with daily supplementation of tocotrienol-rich fraction (TRF) to potentiate anti-tumor immune responses. We had previously reported that DC-vaccine immunotherapy together with TRF supplementation induced protective immunity to tumor challenge. Breast cancer was induced in female BALB/c mice. The mice were randomly assigned into the treatment groups. At autopsy, peripheral blood was collected in heparinized tube and the expression of cell surface molecules  that are crucial for T-cell activation and survival were analyzed by flow cytometry. Tumor was excised from each animal and snap-frozen. Total RNA was extracted from each tumor tissue for microarray and gene expression analysis. Total protein was extracted from tumor tissue for protein expression studies using Western blotting. The results show that systemic administration of 1 mg TRF daily in combination with DC-vaccine immunotherapy (DC + TL + TRF) caused a marked reduction ( Efforts to find a cure for cancer have been ongoing for some time. Researchers have used several approaches, including developing vaccines against some tumors such as breast, lung, colon, and prostate cancers ,4,5,6,7.Tocotrienol-rich fraction (TRF) is the vitamin E fraction extracted from palm oil, which contains 70% mixed tocotrienols and 30% alpha-tocopherol ,23. TocoThe aim of the present study was to investigate the effectiveness of TRF as an adjuvant in enhancing immune response to prevent tumor growth using a syngeneic mouse model of breast cancer to elucidate the mechanism(s) of action using microarray and quantitative PCR approach.Five to eight-week-old inbred female BALB/C mice were purchased from the Experimental Animal Facility of the Institute for Medical Research (IMR), Kuala Lumpur. The animals were housed at the same facility and were given a commercial pellet diet and water ad libitum. The cage bedding was changed every three days. The mice were allowed to acclimatize in their environment and housed under a 12 h light/12 h dark cycle in an air-conditioned room with temperature set at 25 °C. This study was conducted in accordance with international animal ethics guidelines and was approved by the Research and Ethics Committee of the International Medical University (IMU), Kuala Lumpur (Ref: 4.9/55/2007 dated 26 June 2007).2 . At day 5–7, 20 ηg/mL of tumor necrosis factor-alpha  were added to the culture to induce maturation in cells. The DC were usually harvested on day 7–9, depending on their maturation and differentiation, which were monitored using an inverted microscope. The DC were characterized by analyzing the expression of CD11c using flow cytometry  Biosciences, San Jose, CA, USA)The bone marrow (BM) was harvested under aseptic conditions. The tibia and femur bones were obtained from euthanized BALB/c mice and the BM cells were obtained by flushing the cut tibia and femur bones with complete medium using a 22 cc needle syringe as described previously ,24. CompThe tumor cell line used in this study was the 4T1 murine mammary cancer cells, purchased from the American Type Culture Collection (ATCC) . The 4T1 murine mammary cancer cells are spontaneously metastatic tumor cells from mammary gland tumor of BALB/C mice which are reported to be comparable to human stage IV breast cancer . The 4T12 . The next day the 4T1 cells were collected into a 15-mL tube  and harvested by centrifugation (1000 rpm for 5 min). The supernatant was discarded and the cells were resuspended in 1 mL culture medium. Tumor lysate (TL) from the 4T1 cells was prepared by subjecting these cells to several freeze–thaw cycles. The cells were first frozen in liquid nitrogen and then rapidly thawed at 65 °C. The freeze–thaw cycle was repeated 3–5 times. The cell lysates was centrifuged (2000 rpm for 5 min) and the supernatant was passed through a 30-μm nylon filter-column before it was aliquoted in vials and stored at −80 °C until use.The 4T1 cells were cultured in the presence of 8 μg/mL TRF in T25 flasks overnight at 37 °C in a humidified incubator with 5% CO2 for 24 h. Following this, culture supernatant was removed and the cells were washed thrice in Hanks’ Balanced Salt Solution (HBSS). The tumor-pulsed DC were recovered by centrifugation (2000 rpm for 3 min) and resuspended in 1 mL complete medium and left on ice prior to injection into mice.The spent culture medium from DC in a T25 flask was replaced with fresh medium and TL was added. The amount of TL added was based on a 3:1 ratio of confluent DC:TL. The flask was incubated at 37 °C in a humidified incubator with 5% COThe six-week-old BALB/c mice were randomly assigned in to one of the five groups  used in this study . Each grg for 5 min at 4 °C). The supernatant was discarded and the PBS wash step was repeated twice. Following this, the cells were stained with fluorochrome-conjugated antibodies against some mouse antigens, such as CD40-FITC , CD80-PE , CD83-FITC , and CD86-PE , for 30 min on ice. Then, the cells were washed with buffer ) and recovered by centrifugation (1800 rpm for 5 min). The cells were fixed by addition of 500 µL of wash buffer, followed by 500 µL of fixing solution . Each sample was prepared with unstained cells for comparison with stained cells. The tubes were thoroughly mixed before they were analyzed using flow cytometry using the Cell-Quest software provided by the manufacturer  Biosciences, San Jose, CA, USA). The population of interest was gated using the forward scatter (FSC) and side scatter (SSC) dot plot. For each acquisition, 10,000 events were acquired for data analysis. The acquired data was analyzed using the Cell-Quest software. For each sample, the percentages of cells stained with the fluorochrome-conjugated antibodies (FL1 (FITC/green fluorescence) versus FL2 (PE/red fluorescence)) in the gated population were obtained. The changes in the FSC and SSC channel were adjusted and compensated so that the populations were centralized on the dot plot.Whole blood (500–800 µL) was collected into heparin tubes. The red blood cells (RBC) were lysed using a commercial RBC lysis buffer according to the manufacturer-recommended protocol . Briefly, two drops of RBC lysis buffer were added to each tube containing the mice blood. The tubes were incubated at room temperature for 5 min. The lysis reaction was stopped by addition of PBS pH 8.0 solution. The tubes were centrifuged (1000× n = 3) from three groups . Total RNA was also extracted from 4T1 cells for this analysis. Total RNA was extracted using the TRI-reagent solution according to the manufacturer’s instructions . The concentrations of the extracted RNA and ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured using the NanoDrop ND-1000 spectrophotometer . The integrity of the extracted RNA samples was evaluated with the RNA integrity number (RIN) for each sample using the Total RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer . The RIN describes a gradual scale of RNA integrity from 1 (RNA completely degraded) to 10 (RNA without degradation). In general, a RIN that is higher than seven is accepted to be optimal in most of experiments. For the microarray analysis, total RNA was extracted from tumors of mice  database , supplemented with probes derived from the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) set as well as exemplar protein-coding sequences described in the RIKEN FANTOM2 database. The microarray facilities were provided by Malaysian Genome Institute (MGI), University Kebangsaan Malaysia (UKM), Bangi, Malaysia. The complementary RNA (cRNA) synthesis and purification were performed using IlluminaTotalPrep RNA Amplification . All the RNAs were subject to cleanup using RNA cleanup  and hybridization steps followed with the manufacturer’s protocol.SATB1) at the mRNA level. For this experiment, RNA from tumors of three animal groups  and 4T1 cells were used. The RNA samples treated with DNase treatment  were used for the RT-PCR. The reaction mix consisted of 5 µL purified RNA (5 ng), 0.5 µL of enzyme mix, and 12.5 µL of 2 × reaction mix ); 10 µM of reverse and forward primers were added to the microwell plate. Ultra-pure water  was added to each microwell to make the final volume 25 µL. The reaction was run at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 55 °C for 30 s, and an extension phase for 1 cycle at 95 °C for 60 s, 60 °C for 60 s, and 95 °C for 60 s  in a thermocycler . Triplicate samples were run for each RNA. The primers for murine beta-actin (β-actin) and SATB1 genes were purchased from Invitrogen  and R&D Research , respectively. The primer sequences are as follows: (a) β-actin  and (b) SATB1 . Data were normalized relative to the housekeeping gene.In this study, we used quantitative PCR (RT-PCR) to confirm the expression of special AT-rich binding protein 1  for Western blot analysis. Protein concentration of each sample was determined using the Bradford method . Protein extracted (30 ng) from each sample was resolved on 10% SDS-PAGE gels and electro-blotted onto nitrocellulose membrane using a semi-dry transfer system . The membrane was incubated overnight at 4 °C with 5% blocking agent  in wash buffer (0.1% (v/v) Tween 20 in PBS). After the blocking step, membrane was probed with primary antibody (SATB1 or FasL)  for 2 h at room temperature. Subsequently, the membranes were incubated for another two hours at room temperature with a secondary antibody, horseradish peroxidase (HRP) anti-rabbit . Detection was performed using an ECL detection kit according to the manufacturer’s instructions . Following this, the membranes were stripped off from the probed antibodies, blocked, and re-incubated with mouse monoclonal GAPDH  followed by HRP-labeled anti-mouse secondary antibody . Bands on autoradiography films  were quantified using ImageJ software and normalized against GAPDH. The list of primary and the corresponding secondary antibodies used for the Western blotting assay is listed in t-test and SPSS version 20 for the most of data. For the tumor incidence data, statistical analysis was done using the one-way ANOVA with post hoc Tukey test for multiple comparisons (SPSS version 20). The IMAGE J software (http:rsb.info.nih.gov/ij/index.html) was used for the densitometry analysis of protein bands whilst the Bead Studio 2.1 and GENE SPRING 7.0 were used for microarray analysis. Most of the data represent the ± SD of triplicate measurements. The quantitative PCR data represent mean value with ± SEM of the triplicate measurements.Statistical analysis was done using the Student The anti-tumor effect was assessed using a syngeneic mouse model of breast cancer. Previously, we had reported that the combination of DC pulsed with TL (DC + TL) and TRF supplementation showed marked anti-tumor effects, as a marked inhibition of tumor growth and metastasis was observed in the mice that received this treatment ,24. In tp > 0.05) was observed in mice in the DC + TL + TRF groups  in the number of PBL that expressed CD40, CD80, CD83, or CD86 in PBL from the DC + TL + TRF groups compared to the other groups  of mice was determined as these molecules are reported to be important surface markers that provide co-stimulatory signals required for T-cell activation and survival ,25,26. Gr groups . The expr groups . For CD8r groups . For CD8r groups . For CD8r groups .Fas by FasL expressed on the activated CTL [The Fas/Fas ligand (FasL) is reported to be one of the process required for the maintenance of immune privilege ,28,29. Ip < 0.05) or 99% (p < 0.01). For this analysis, we focused on studying the difference in gene expression in tumors from the DC + TL and DC + TL + TRF groups as these treatment groups showed very similar genes expression pattern in the dendogram (p < 0.05) for, there were four genes that were differentially regulated  were down-regulated (p < 0.01), only one gene was differentially regulated  down-regulated in tumors from the DC + TL + TRF group when compared to tumors from the DC + TL groups . In order to reduce non-specific binding errors, the normalization intensity was conducted on all samples and groups. A dendogram a was creendogram a and Conendogram b analysiegulated c. The exegulated . When thegulated c. This gL groups .SATB1 gene in the tumor tissues extracted from the three DC-treated groups  was analyzed using quantitative PCR (qPCR). Total RNA extracted from cultured 4T1 cells was also included in the analysis. A marked (p < 0.05) reduction in the expression of SATB1 gene was observed in tumors extracted from mice treated with DC + TL + TRF (SATB1 was only 0.14-fold (p < 0.05) lower expression of the SATB1 gene when compared to DC alone (0.59) or cultured 4T1 cells (1.0)  .SATB1 protein expression in tumors from the DC + TL + TRF and DC + TL groups when compared to tumors from the control or vehicle groups (SATB1 expression to be reduced in DC + TL + TRF (23.3% of relative density) group compared to other groups  was upregulated, whilst three genes  were down-regulated (p < 0.01) was used, only one gene (SATB1) was found in this category  in tumors from mice treated with DC + TL + TRF , which are implicated in the loop domain organization of chromatin or chromatin remodeling [SATB1 gene [p < 0.0001) with prognostic outcomes of cancer patients [SATB1 gene was able to reprogram chromatin organization and the transcription profiles of breast tumors, which promoted cancer growth and metastasis [Microarray analysis performed to compare gene expression in tumors from DC + TL and DC + TL + TRF flagged four differentially expressed genes. One gene (egulated  when thecategory c. The SATL + TRF . This SAi et al.  reportedi et al. ,39. In ty in TH2 ,39,40. Tmodeling ,39,40. ISATB1 gene correlated well with the protein expression in tumors excised from the experimental animals. As observed with the gene expression studies, there was marked suppression of SATB1 protein expression in tumors from mice treated with DC + TL + TRF (The expression pattern of the TL + TRF .SATB1 was identified to be one of the potential targets that could have produced some of the anti-tumors effects observed. Further studies are being planned to elucidate the role of the SATB1 gene in tumor growth and spread as well as to understand how TRF is able to modulate the expression of this gene.In conclusion, this study has shown that TRF can be used to supplement DC-vaccine immunotherapy using an immunocompetent experimental model. The DC + TL + TRF approach appears to induce activation and promote survival of T-cells, as judged by the increased expression of CD40, CD80, CD83, and CD86 in PBL as well as FasL in the tumors. Increased expression of FasL protein in tumor tissue from the DC + TL + TRF-treated mice suggest that CTL activation may have taken place and this could have contributed to inhibition of tumor growth. One of the limitations of this study is the small number of mice used for the study. Also, the scope of the present study is rather narrow as we were not able to include more parameters to investigate. However, despite this the results are significant and we have managed to identify a key target protein that we could do more work on.


Skull fractures are common injuries in young children, typically caused by accidental falls and child abuse. The paucity of detailed biomechanical data from real-world trauma in children has hampered development of biomechanical thresholds for skull fracture in infants. The objectives of this study were to identify biomechanical metrics to predict skull fracture, determine threshold values associated with fracture, and develop skull fracture risk curves for low-height falls in infants. To achieve these objectives, we utilized an integrated approach consisting of case evaluation, anthropomorphic reconstruction, and finite element simulation. Four biomechanical candidates for predicting skull fracture were identified  and evaluated against well-witnessed falls in infants (0–6 months). Among the predictor candidates, first principal stress and strain correlated best with the occurrence of parietal skull fracture. The principal stress and strain thresholds associated with 50 and 95% probability of parietal skull fracture were 25.229 and 36.015 MPa and 0.0464 and 0.0699, respectively. Risk curves using these predictors determined that infant falls from 0.3 m had a low probability (0–54%) to result in parietal skull fracture, particularly with carpet impact (0–1%). Head-first falls from 0.9 m had a high probability of fracture (86–100%) for concrete impact and a moderate probability (34–81%) for carpet impact. Probabilities of fracture in 0.6 m falls were dependent on impact surface. Occipital impacts from 0.9 m onto the concrete also had the potential (27–90% probability) to generate parietal skull fracture. These data represent a multi-faceted biomechanical assessment of infant skull fracture risk and can assist in the differential diagnosis for head trauma in children.The online version of this article (10.1007/s00414-018-1918-1) contains supplementary material, which is available to authorized users. Falls are the leading causes of nonfatal, unintentional injuries in infants ≤ 1 year of age . HistoriGiven ethical restrictions for conducting controlled falls in children, pediatric biomechanics of falls are investigated using case reports , 5, retrTo improve on the limitations of the individual study types, laboratory-based studies are often paired with FE model simulations to identify fracture thresholds. Roth et al. , 20 and In this study, we utilized an integrated approach combining case-study evaluation, full-body anthropomorphic infant fall reconstruction, and FE simulation to identify the skull fracture risk associated with low-height falls in infants. The infant head FE model incorporates material properties of the human infant skull and suture from children over the age range of 19 days to 4.5 months . Using mTo identify biomechanical response metrics and thresholds that are predictive of parietal skull fracture, data from simple well-witnessed falls with detailed event descriptions and radiological reports were examined. The study was approved by the Institutional Review Board of the University of Pennsylvania and Children’s Hospital of Philadelphia (CHOP) and carried out in accordance with the IRB approved guidelines and regulations. All infants less than 6 months of age with a history of a fall  admitted to the CHOP between June 2006 and September 2009 were evaluated as potential subjects. To be considered for the study, the medical history had to state that the fall event had been witnessed by two adults, one adult and one child old enough to communicate, or one adult whose statement had been designated as without suspicion of abuse by the CHOP child protection team. When the incident was witnessed by two or more persons, the incident description was reviewed carefully to ensure that there was no inconsistency in the details of the incident history. Inclusion criteria also specified that CT and/or MRI scans were obtained within the first week of hospitalization and evaluated by a radiologist. Exclusion criteria were a history of child abuse, mental retardation, drug dependency, birth complications, hemophilia, and pre-existing skull or brain abnormalities . Cases with a history of a complex fall that would be difficult to reconstruct , and those with ambiguous event descriptions were also excluded. Cases with fall heights greater than 5 ft were excluded due to limits of the test facility. Of 263 cases screened, 44 cases met the inclusion/exclusion criteria. Cases were further evaluated to determine whether the details of the incidents required for reconstruction were explained clearly in the medical records. If needed, care providers and/or parents were contacted following approved consent procedures to obtain additional event information. The required details were the position of child immediately prior to and after the fall, an estimation of the height of the fall, type of flooring that child landed on, the first part of the child’s body to come into contact with the ground, anything else the child came into contact with during or after the fall, the height of the person involved in the fall, and the age of the witnesses. Additional details about the sustained injuries and health conditions of the child prior to the accident were also gathered. Twenty-six of the 44 cases had unambiguous, well-described fall histories. These 26 patient records and images were reviewed carefully by a radiologist to document; if possible, the exact location of skull fracture or other physical signs of head impact, such as soft-tissue swelling or subgaleal hematoma. Cases were excluded from further consideration if images were of poor quality or if no physical signs of head impact were observed. Cases were excluded if there was a lapse of more than 1 day between accident and hospital visit, thus decreasing the efficacy of using the CT/MRI data to determine exact impact location. Based on the medical record, witness and/or interview report, and radiology report, 11 of the 26 subjects were enrolled in the study: 7 with diagnosed parietal skull fracture  and 4 with no skull fracture. Details of each case, including a brief description of the event, age, sex, estimated fall height, impact surface, area of impact, and skull fracture are provided (Table E = 535 ± 139 MPa) attached together with a silicone material (E = 2.1 ± 0.2 MPa) and covered with a 1-mm-thick latex cap that duplicate the mechanical response of the infant skull, suture, and scalp reported in the literature [G = 765 ± 44 Pa) similar to infant brain. There was no air space in the surrogate head. The mass of the body was also adjusted so the overall surrogate mass matched the overall child’s body mass. Additional details on the biofidelity and validation of the surrogate are reported in [To estimate the head impact force of each child, the 11 cases were reconstructed using a whole-body biofidelic anthropomorphic infant surrogate . A six dterature . The surterature . For eacterature , and theorted in .The contact surfaces for the 11 cases were concrete, tile, linoleum, laminate, or carpet with carpet pad. The rigid metal surface of the heavy force plate was used as the closest approximation to concrete and tile. Examples of the other three impact surface materials as stated in the history report were obtained and clamped to the force plate. For each case, the primary impact location was determined from the fall description, medical history, and the radiological reports of fracture and soft-tissue swelling. For fall reconstructions, the fall height, flooring, identified head impact location, and the fall description from the medical and witness reports for each case were matched and verified via a high-speed digital video . Carbon paper was used to mark impact location on the surrogate head. The nature of accident reconstruction entails some uncertainties, and there is trial-to-trial variability, so it is impossible to determine with absolute certainty the forces applied to the head during the impacts. Creating a corridor of possible responses by performing multiple trials is a way to take these uncertainties into consideration in real-world accident reconstruction studies , 34. Theμ0; strain-sensitive nonlinear characteristic parameter, α; Poisson’s ratio, υ; the relaxation moduli, C1 and C2; and time constants, τ1 and τ2, in the Prony series  . The infbase1/3) . Becausebase1/3) . Specifientclass1pt{minimabase1/3) . The braentclass1pt{minima−3 m/s to 2224 N. Depending on available surface area, 5–24 locations on each surface were tested. Tests near boundaries of the flooring sample were excluded. An average stress-strain curve for each material was implemented in the material evaluator of ABAQUS . Linoleum and laminate were represented with second-order-reduced polynomial models. Carpet and carpet pad were modeled, respectively, with second-order and first-order forms of ABAQUS’s hyperfoam material model. The concrete and tile impact surfaces were represented as rigid bodies. Table ρ) and coefficients for each flooring surface.To model the impact surfaces in the simulations, the linoleum, laminate, carpet, and carpet pad were indented with a 0.076-m platen at 0.02 × 10N = 33) in ABAQUS explicit. For each FE simulation, the initial velocity of the head was adjusted until the simulated impact force-time history matched the peak and duration of experimental impact force trace within 5% error tolerance  is the probability of parietal skull fracture for the given value x of the predictor candidate. Variables a and b are the regression coefficients. The quality of fit for the risk curves was then evaluated for the four candidate parameters using the Nagelkerke R2 and Cox and Snell R2 statistics to determine the parameter with the highest correlation with skull fracture. Receiver operating characteristic (ROC) curves were also generated, and the area under the ROC curves (AUROC) was used as another measure of how well each parameter distinguished between parietal bones with and without skull fracture. For each candidate fracture parameter, the threshold values corresponding to the 50 and 95% likelihood of skull fracture were extracted from the logistic regression, and the optimal ROC threshold (optimizing specificity and sensitivity) extracted from the ROC curve. For each candidate, the Q2 (or median) risk curve and associated skull fracture threshold value were used for further analysis in this study. The Q1 and Q3 risk curves, and associated threshold values, were used to demonstrate the range of potential uncertainties or errors in injury risk curve development using our real-world accident reconstruction approach.Each of the 33 simulations generated two peak values for the parietal skull plate  for all 4 candidate predictors. For each of the 4 candidate skull fracture parameters, the 66 generated data points were categorized into one of the three simulation groups representing the first, second, and third peak head impact force quartiles (Q1-Q2-Q3) measured in the reconstruction experiments. For each simulation group, a binary classifier was assigned to each response data point to designate the presence (1) or absence (0) of fracture, based on the radiological report. Binary logistic regression analyses were then performed, and Q1, Q2, and Q3 parietal skull fracture risk curves were created  with distribution functions of the following form:N = 10 drops/condition). The trials producing the minimum, median, and maximum head impact forces for each height-surface-location combination were selected for FE analysis in this study , and Cox and Snell R2 (0.519 and 0.516). The threshold values corresponding to the 50 and 95% probabilities of parietal skull fracture, as determined from the developed injury risk curves for the four predictors are provided in Table For each potential skull fracture predictor, 66 data points were extracted from the FE simulations . Each data point was assigned a designation of presence of parietal fracture or absence and is displayed in Fig. SThe peak first principal stress and strain associated with the minimum, median, and maximum head impact force from low-height falls is given in Table SClinical differentiation of accidental and abusive head injury etiologies has proven to be a persistent problem for physicians. Given the paucity of documented pediatric accidental cases in the literature, the 11 well-witnessed, real-world infant falls in our clinical dataset, along with kinematics estimated from anthropomorphic surrogate event reconstructions and biomechanical parameters from FE simulations, provide the biomechanics community with valuable data that can facilitate validation of future computational models of infant head injuries. In addition, kinematics measured in our controlled surrogate drops and biomechanical parameters from corresponding FE simulations representing fall scenarios involving three heights, two surfaces, and two head impact locations, can serve as a data set for future studies investigating the likelihood of head injury in low-height falls.To develop skull fracture risk curves, we used anthropomorphic surrogate studies and multiple biomechanical response parameters from FE simulations. Potential of using these biomechanical parameters to be used as skull fracture predictors in infants were assessed not only in terms of quality of fit of the corresponding developed risk curves and their prediction accuracy rates but also by comparing the distribution of these parameters with the actual location and pattern of skull fracture in real-world falls. An additional large set of controlled head-first fall drop tests  were useUsing surrogate and FE reconstructions of real-world fall accidents, we found skull fracture thresholds similar to failure stress and strain values reported for infant parietal skull . Our 50 When placed in context with ultimate stresses reported via three- and four-point bending of pediatric crania of varying ages, our first principal stress fracture threshold (25.229–36.015 MPa) and Coats and Margulies’ ultimate stress 30.95 MPa) underscore the order of magnitude range in skull fracture thresholds for ultimate stress over the age spectrum from infant to adults, with ultimate stress increasing dramatically during the first year, then asymptoting to adulthood Fig. . Marguli.95 MPa u−1, respectively. Of note, only Motherway et al. [Several studies have tested the adult skull in tension or compression. Evans and Lissner  reportedy et al.  performey et al.  and Daviy et al. , respectIn both the pediatric and adult literature, ultimate strain measurements are surprisingly sparse. Wang et al.  reportedIn addition, in all simulated fall scenarios, we identify consistent predictions for parietal fracture likelihood and fracture pattern using first principal stress and strain as skull fracture metrics. Coats et al.  used firTo date, the present study represents the first FE model to assess likelihood of infant skull fracture in a variety of low-height fall scenarios with the aid of injury criteria developed based on real-world fall cases and validated against ultimate stress and strain values derived from mechanical tests on infant skull cadavers. Previously, we reported that peak head impact forces for 0.9 m falls onto carpet approach those for concrete, but peak head impact forces for 0.6 m falls onto carpet were significantly lower than those for concrete . In the Examining published clinical studies on pediatric head injuries following low-height falls, we focus on those limited to young children and infants. Among the children ≤ 2 years old who experienced non-inflicted head injuries in Duhaime et al.’s  prospectBoth first principal stress and strain predictors in our study indicate probabilities of parietal fracture for 0.9 m falls onto concrete with occipital impact. The bone-suture junction is less structurally rigid than the skull plate in infants  that makStudies published nearly 70 years ago reported fractures on the external skull surface in regions of outward bending and on the internal surface in regions of inward bending. While inward bending always occurred under the site of impact, outward bending sometimes occurred at considerable distances away from the site of impact , 47. GivAlthough this study significantly advances pediatric head trauma literature by proposing new biomechanical fracture criteria and predicting parietal fracture in low-height fall scenarios, the limitation of our study is the lack of any occipital fractures in our clinical data set. As a consequence, we were unable to develop injury criteria for occipital fracture or to predict occipital fracture in fall scenarios. However, making the assumption that principal stress and strain fracture thresholds are similar for parietal and occipital skull plates, we would predict a low probability of fracture for the three occipital impact cases in Table One additional important consideration with our parietal skull fracture risk curves is that a sideways fall often concludes with shoulder or limb contact before the head. Our whole-body surrogate reconstructions of short falls simulated worst-case risk scenarios with head-first contact. Thus, the likelihood of skull fracture risk might differ from those reported here if the fall were “broken” by contact of another body part during the fall.We developed parietal skull fracture risk curves for infants under 5.5 months old through reconstruction of real-world accidental falls in infants. To minimize the uncertainties accompanied by accident reconstruction procedure, rigorous inclusion criteria were employed during data collection process, impact kinematics were estimated through whole-body anthropomorphic surrogate reconstructions, and precise and comprehensive approaches were used to verify the reliability and precision of surrogate and FE reconstructions. FE simulations were validated against not only the peak impact forces but also the entire impact force-time histories measured in surrogate reconstruction drops. As an improvement over the existing accident reconstruction studies, FE simulations were also verified by considering stress and strain distribution in terms of the location and pattern of skull fracture. Among all four potential predictors, maximal first principal stress and strain best correlated with the occurrence of parietal skull fracture, and their corresponding proposed tolerances are in agreement with published in vitro material failure tests. Finally, the resulting risk curves were used to evaluate the worst-case likelihood of parietal skull fracture in head-first, low-height infant falls. We conclude that the likelihood of parietal skull fracture in head-first falls from 0.3 m is very low, regardless of fall condition. Falling from 0.9 m onto the concrete on either occipital or parietal site can result in parietal skull fracture. An improvement over anecdotal clinical reports and heuristic evidence, the outcomes of the present study can be used to more accurately assess likelihood of skull fracture in infant falls for a variety of fall heights, impact locations, and contact surfaces.ESM 1(DOCX 15 kb)ESM 2(PNG 518 kb)High resolution image (EPS 205668 kb)


Modern mass spectrometry, including electrospray and MALDI, is applied for analysis and structure elucidation of carbohydrates. Cyclic oligosaccharides isolated from different sources (bacteria and plants) have been known for decades and some of them (cyclodextrins and their derivatives) are widely used in drug design, as food additives, in the construction of nanomaterials, etc. The peculiarities of the first- and second-order mass spectra of cyclic oligosaccharides  are discussed in this minireview. The most studied of them, cyclodextrins  by mass spectrometry alone is impossible (because of m/z = nm/nz), but they are separable by ion mobility spectrometry (IMS) due to their difference in size and shape + in a positive ion mode.In MALDI MS, CDs give only singly charged ions. For example, for permethylated β-CD in DHB matrix, only [M + Na]ion mode . Howeverion mode . It was 4), heptakis-β-(2→1)-fructofuranose , octakis-β-(2→1)-fructofuranose   2+ “fragment by glycosidic cleavages giving B-series of ions...”  in the table of the MS2m/z data and presented a description of them below the table. Briefly, A-series is formed by doubly charged ions differed in 81 Th. B-series arises due to charge separation reaction due to loss of [Glcn + H]+ (difference in 162 Da). Low abundant C-series is formed by singly charged ions smaller than B-peaks on 264 Da; this cleavage is accompanied by C-H hydrogen transfer (demonstrated by H/D isotopic exchange in OH groups). Low abundant D-series may be regarded as a result of loss of H2O from B-series ions . For a representative MS2 spectrum, see It is impossible to find out “reducing” and “non-reducing” ends for a cyclic oligosaccharide molecule , so an attempt to apply the generally accepted Domon-Costello nomenclature of oligosaccharide fragmentation  leads toaimed in ), thoughaimed in : “...we aimed in  (p. 1568aimed in  was refeaimed in  introduc2O (ca. 18 Da), for permethylated OSs, C2H6O (ca. 46 Da), for peracetylated OSs, C4H6O3 (ca. 102 Da), etc. Note, that the MS of acyclic OS having an anhydro unit in its composition looks like that of cyclic one due to their isomerism. Some structural studies based on MS  are presented below. A previously unknown glucan  was isolated from a recombinant strain of a Rhizobium meliloti TY7, an ndvB mutant carrying a locus specifying β- glucan synthesis from Bradyrhizobium japonicum USDA110, using reversed phase chromatography +  and [M + Na]+ , respectively (M = Glc12). Acyclic dodecahexaose should be heavier by 18 Da, so, this one had likely a cyclic structure. 1H- and 13C-NMR spectra were rather complex, but they demonstrated the presence of mainly β-(1→3)-linked (definitely not of α-configuration) glucose residues along with a minor β-(1→6)-glycosidic bond. The first Smith degradation afforded the product consisting of eleven hexose units . The second Smith degradation gave the product consisting of only β-(1→3)-Glcp units according to 1H- and 13C-NMR spectra -β-glucosyl), which was resistant to the next Smith degradation. Surprisingly, the MS of this carbohydrate expected to be cyclodecalaminarinose exhibited the main peak at m/z 1735, which the authors assigned it to [M + 5Na]+, which is definitely unbelievable. A more reasonable explanation of this peak is complexation of the analyte with glycerol usually used as a FAB matrix , for [Hex10 + Gro + Na]+, calculation gives m/z 1735.56, so the structure of the starting material was assigned to β-(1→6)-laminaro(cyclodekakis-β-(1→3)-glucosyl). Later, the same authors isolated the both cyclic OSs  from different mutants of Bradyrhizobium japonicum + were mentioned with no data. Heterogeneity of these cycles (the presence of one β-(1→6)-Glcp units inside each cycle) was approved by NMR spectrometry  and molecular dynamics calculations. Cyclic OPGs usually bear side chains, e.g., phosphatidylglycerols giving a +C3H7O5P increment (ca. 154 Da each) +, calcd. m/z 2792.8 , m/z 2969.1, [M2 – 2H + 3Na]+, calcd. m/z 2968.80. In ESI MS, the main triply and doubly charged ions were observed: m/z 915.9, [M1 – 3H]3− ; m/z 967.7 [M2 − 3H]3−, ; m/z 1374.6, [M1 − 2H]2− , m/z 1451.6, [M2 − 2H]2− . There is no explanation why the exp./calcd. data coincidence for ESI MS was not so good as for MALDI TOF MS, possibly, this due to low resolution of the triple quadrupole analyzer used in this work. No MS2 data were reported.Similar studies were carried out for OPGs from several agriculturally important microorganisms  . In this3 in DMF, and the purified amine was acylated with biotinamidohexanoic acid N-hydroxysuccinimide ester. MALDI TOF MS (DHB matrix) of monoaminated and monobiotinylated cyclooligosophoraoses displayed prominent [M + Na]+ peaks for d.p. 17 to 22 which showed good coincidence with the calculated values. As expected, the difference between the corresponding peaks with the same d.p. is equal to ca. 339 Da .The appropriate side chain  can be introduced artificially. Biotinylation through amidocaproyl spacer was done by Cho et al. . A mixtu+, K+, and Cs+ (not H+) in MALDI MS were much higher that of corresponding ionized maltohexaose and maltoheptaose, respectively; differences showed an increasing trend with the radii of metal cations, and β-CD had higher ionization efficiency than α-CD. However, no proofs of general character of this regularity were demonstrated and even if such proofs will be found, it seems problematic to use this phenomenon in assignment of isomeric acyclic and cyclic OSs without authentic or reference samples  between linear and cyclic oligosaccharides. In fact it was shown by Choi et al.  that theles (cf. ).To use fragmentation techniques to distinguish linear and cyclic OSs is promising in order to avoid isolation of individual compounds. The first attempt to realize this approach was done by P.J. Derrick and colleagues . Dextran+ and [M + H + K]2+ ions were fragmented for each compound. It was shown that three methods of activation are complementary. For CDs, all of the fragments were formed by glycoside bonds rupture, no cleavages of residues were observed. Comparison of CE50 demonstrated that slightly higher collision energy is needed to cleave CD than the related linear maltooligosaccharide . Though the authors claim that CID/HCD combination opens a way for deciphering of structures of complex carbohydrates “alone or in mixture”, we do not share their optimism because the observed effects seem very tiny.Later, a similar regularity was demonstrated for ESI MS in more sophisticated experiment using CID, PQD, and HCD activation procedures for three pairs of α-CD/maltohexaose, β-CD/maltoheptaose and γ-CD/maltooctaose . A LTQ oThus, the choice between acyclic or cyclic structure can be supported by ESI or MALDI MS if a small amount (enough for an NMR study) of rather pure compound is isolated, but such a choice for a component in a complex mixture  remains problematic despite the great progress of tandem techniques during the last thirty years.2 [n + H]+, n = 2–5 by activation of the protonated complex .At present time, there are various derivatives of cyclic OSs, especially CDs prepared for different purposes. First-order MS  can obviously applied for profiling and determination of d.s. ,37. SincO-6-tosylation of Glcp unit + and [M + Na]+ were observed in MALDI and ESI MS. LID and CID of the protonated molecule resulted in similar secondary-order mass spectra with predominant formation of aminohexose-containing fragments, so, a proton is definitely localized on the amino group and the first cleavage of glycosidic bond occurs near the HexN residue  and bis(6-O-tosyl)-β-CD (12–14). One can assume that mass spectra of decay for regioisomers of modified CDs (as well as for any cyclic oligosaccharides) may differ from each other since a double rupture of glycosidic bonds can result in different combinations of primarily bonded units. A critical consideration of this approach may be formulated in two questions:(1)Are the bonds between substituents and the carbohydrate units strong enough in comparison to glycosidic bonds, and(2)Do transitions of substituents between carbohydrate residues  take place?The influence of the positions of regioisomeric, disubstituted β-cyclodextrins on their ESI MSudied in  for two 9a, in AC, they are separated with one and four unsubstituted units, 10a, and in AD, 11a, substituted units are separated with two and three unsubstituted units, 2 for all three [M + H]+ ions , but it is absent in MC2 of AD and AC. The fragment consisting of two substituted and one unsubstituted unit (m/z 963) is absent в MS2 of AD. The peak of the ion resulting from elimination of one substituent (m/z 1732.5) is present in MS2 of all three regioisomers, nevertheless, this pathway does not dominate. For bis-6-O-tosylates, the fragmentation differs drastically: CID MS2 of [M + H]+ for ditosylates AC and AD are practically the same, and for AB, the same pattern of ions was observed though peaks assigned to ditosylated maltose (m/z 633) and ditosylated maltotriose (m/z 795) have higher relative abundances than those of AC and AD  followed by NHMe2 loss. The [M − (NMe2R)]2+ ion eliminated one singly charged Glc residue from β-CD thus opened the cycle. Two pathways of further fragmentation were realized. Path 1: Glc neutrals are sequentially lost. Path 2: the second alkyl and NHMe2 were lost followed by sequential loss of Glc units. Cleavage of the carbohydrate residues also occurred simultaneously. This complicated scheme of thoroughly investigated fragmentation was supported using MS3 to reveal fragment ion transitions . All studied compounds had similar fragmentations, no drastic differences between O- and N-succinates were reported.Cyclic OSs, especially CDs, are used as starting compounds for the construction of complex organic and hybrid molecular systems. For example, CDs are applied for preparation of drug delivery agents in which a CD core is tethered by ether or ester bonds with linkers bearing side ionogenic groups. A scheme of CID fragmentation of a series of surfactants based on 6-ted β-CD  was pres+ were also identified. It was stressed that “LC with on-line ESI MS detection has problems with the analysis of polymer species related to formation of multicharged ions thus creating difficulties in spectra interpretation, especially in the case of polydisperse oligomer mixtures” + and [M + Na]+ were present, and their MS2 spectra differed drastically. For MS2 of [M + H]+ (c.e. 20 eV), stepwise losses of HOAc and Glc units were observed: after elimination of four HOAc molecules, elimination of one Glc unit occurred until all co-monomer units were depleted. This process was readily illustrated in the table of all possible m/z values, where really observed m/z ones were highlighted forming a kind of ladder. For MS2 of [M + Na]+ (c.e. 120 eV), only consecutive losses of four molecules of HOAc  were observed. In a small m/z range, abundant peaks corresponding to acetylated Glc unit fragments were present. As it was demonstrated previously by NMR and MS [2 alone that there were no oligomeric hydroxybutyryl substituents (which may occur due to esterification of a hydroxyl to give a hydroxybutyryl moiety). Abundant [M + Na]+ ions along with quite visible [M + H]+ ones were observed for each component in ESI MS. For MS2 of [M + H]+ of 5HB-substituted β-CD (c.e. 15 eV), a subsequent loss of Glc units (162 Da) accompanied by losses of HB substituents (104 Da) and their anhydro forms (86 Da) occurred. A similar "ladder", but with smaller steps was pictured in the table created analogously to MS2 of TABCD. MS2 of the [M + Na]+ ion (c.e. 110 eV) contained peaks corresponding to different fragmentation pathways. Ions m/z 1483 and m/z 1379 are formed due to losses of one and two HB molecules. The ion m/z 1425 resulted in loss of one Glc unit, and the ion m/z 1339 was formed due to (Glc-HB) loss (104 + 86 Da). Because of the ion m/z 1397  was not observed, one can conclude that there were likely no di-HB substituent attached to β-CD. The origin of the quite abundant m/z 1543 ion (loss of 44 Da) was not definitely explained by the authors; due to low resolution, it was impossible to find out what neutral (C2H4O or CO2) was eliminated. The above fragmentation pathways classified by Peptu et al. are presented in For the last two decades, CDs were used as scaffolds for star polymers, the best known being the oligolactide-tethered CDs produced by ring-opening polymerization of lactide (the cyclic dimer of lactic acid) initiated by CD, where oligolactide forms a chain attached to the CD ,62,63,64ixtures” ,66. Totaixtures” , and β-CR and MS , the app2 and MALDI LID) were thoroughly described for the [β-CD-LA4 + Na]+ ion with total assignment of composition of fragment ions. In contrast to the above examples, multiply repeated units in substituents capable of fragmentation were definitely present, so three other pathways were observed designated as G (loss of substituted Glc unit) and E1 and E2 (fragmentation of side lactide chains), + and Li+ adducts; heavier ions having more lactate units gave homologous fragmentation patterns.Recently, a mixture of derivatized β-CDs partially substituted with oligolactide chains was studied by GPC, NMR, and MS . From thO-benzylated (with only two free OH groups) β-CD was transferred to a homologous mixture of oligopolylactides, which was characterized by MALDI TOF profiling along with NMR spectrometry +, [M + 2H]2+, [M + Na]+, [M + K]+, and a moderate peak of an [M − H]− anion. The ions [M + NH4]+ are formed only for cyclic N-acetylglucosamines 19b–24b, not for cyclooligoglucosamines 19a–24a.First-order, electrospray ionization (ESI) mass spectra of compounds 2 spectra of cyclooligo-β-(1→6)-d-glucosamines 19a–24a and cyclooligo-β-(1→6)-d-N-acetylglucosamines 19b–24b, cleavages of the glycosidic bonds were the principal processes, they are accompanied by eliminations of molecules of water (and ammonia for cyclooligo-β-(1→6)-d-glucosamines) [19a produced the [M + H]+ and [M + 2H]2+ ions. The main fragment of activation (c.e. 25 eV) of the singly charged ion, [GlcN + H]+  was described as a result of rearrangement. Peak at m/z 203.1023 . The peaks at m/z 185.0919, [GlcN2 + H − C4H8O4 − H2O]+ and m/z 167.0814 [GlcN2 + H − C4H8O4 − 2H2O]+ was explained analogously . The peak at m/z 180.0861 was assigned to the [GlcN + H + H2O]+ ion. The origin of peaks at m/z 174.0756 and m/z 186.0761  was difficult to understand, possibly they occurred due to unknown skeletal rearrangements different from those known before  [m/z 186 and m/z 168 were formed due to the transfer of the CH2CO fragment from one unit to the amino group of the other one followed by cleavage of the glycosidic bond and the loss of one or two molecules of water . Activation of the [M + 2H]2+ ion (c.e 10 eV) resulted in appearance of the main peak at the same m/z, but differed from the residual signal by the position of isotopic peaks (see above) along with smaller peaks of the [GlcN + H − H2O]+ and [GlcN + H − 2H2O]+ ions  and the small peak at m/z 204.0871 . The CID MS2 of the [M + Na]+ ion was not described due to its low intensity.In the positive ion mode ESI CID MSsamines) . For exacN + H]+  had the residue) . In thisO]+ ions . The pea−. As an example, the CID MS2 (c.e. 20 eV) of the ion [M − H] − of 20a was considered. The decay of this anion differed from that of protonated molecule because the main fission occurred not at glycosidic bonds. The small peak at m/z 160.0612 can be considered as a deprotonated link of glucosamine [GlcN − H]−, i.e., the product of cleavage of two glycosidic bonds, nevertheless, the ion [GlcN2 − H]− was not observed. The ions of lower masses occurred due to elimination of molecules of ammonia  and water , and the ion at m/z 101.0247 (C4H5O3−) was possibly formed by elimination of the neutral C2H5NO moiety that may correspond to the O(1)-C(1)-C(2)-N(2) fragment. The main fragment peak at m/z 220.0831 (C8H14NO6−) can be formed only as a result of a cleavage of the C—C bond (presumably C(4)-C(5) and C(5)-O(5) of one of the residues with the retention of one (1→6)-glycosidic bond and the rupture of another one). Small peaks at m/z 464.1906 and m/z 446.1797 corresponded to elimination of one or two molecules of water. The peak at m/z 423.1628 (C16H27N2O11–) may be a result of elimination of the O(1)-C(1)-C(2)-N(2) fragment of one unit. The further elimination of the C2H4O2 fragment resulted in formation of m/z 363.1435 (C14H23N2O9−) anion. The ion at m/z 280.1053 (C10H18NO8−) corresponded to the formal addition of the C4H8O4 fragment to the deprotonated glucosamine residue. Formation of this ion was proposed through the rupture of C-C and C-O bonds in two glucosamine residues with the retention of the third one as a core. It was concluded that primary fragmentation of the negatively charged ions (deprotonated molecules) proceeds in a different way than that of positively charged ones [Mass spectra of the negatively charged ions of the studied compounds demonstrated low abundant deprotonated molecules [M − H]ged ones .-N-acetylglucosamines were similar to those of their acyclic analogs. For example, MS of cyclic dimer of N-acetylglucosamine 19b possessed peaks of [M + H]+, [M + NH4]+, [M + Na]+, and [M + K]+ ions. CID MS2 of [M + H]+ ion resulted in a cleavage of glycosidic bonds and formation of the ion at m/z 204.0865 ([GlcNAc + H]+); under c.e. of 10, 15, and 20 eV, the peak of this ion was the most intense. Elimination of one, two, and three molecules of water resulted in formation of ions at m/z 186.0761, m/z 168.0655, and m/z 150.0550, respectively. At c.e. 40 eV, the peak at m/z 138.0549  became the main one. Two other intense peaks (c.e. 40 eV) at m/z 144.0654 (C6H10NO3) and m/z 126.0549 (C6H8NO2) were assigned to the fragment ions [GlcNAc + H − C2H4O2]+ and [GlcNAc + H − C2H4O2 − H2O]+, respectively. Fragmentation of the ion [M + NH4]+ (c.e. 20 eV) was similar to that of [M + H]+. In the range of higher m/z, the products of elimination of one ant two molecules of water, m/z 389.1544 and m/z 371.1451 were found. In CID MS2 (c.e 50 eV) of metallated molecules of 19b, only cleavages of two glycosidic bonds were revealed , and elimination of H2O from these fragments resulted in [GlcNAc + Na − H2O]+, m/z 208.0575 and [GlcNAc + K − H2O]+, m/z 224.0247. For the [M + Na]+ ion, elimination of one and two molecules of water was observed. For cyclic oligomers having from three to seven GlcN units, the same losses were observed.Mass spectra of cyclic oligo2 of protonated molecules of isomers 25 and 26 was claimed in [2 of their [M + H]+ ions  contained the same signals with slight difference in relative abundance. A small peak at m/z 323 related to the disaccharide fragment [GlcGlcN + H]+ was observed for both oligosaccharides .The aim to reveal characteristic differences in the CID MSaimed in . The pos2 of the doubly charged ions [M + 2H]2+  were much more prominent : for 25, its relative abundance is 0.6 %, whereas for 26 it is equal to 33 %, so, in more than 50 times higher 2+ of 25 and 26 contained only peaks of glucosamine unit and its fragmentation products.At the same time, the differences in CID MSrominent a,b. The n of 21a c gave anshown in  for aminA critical review of traditional methods and recent achievements in the analysis of cyclodextrins and their derivatives is presented in . This paN-succinylamido linker was done by high-resolution positive and negative ESI MS + was presented and described. Successive losses of HBr (80/82 Da) and C6H5CO2H (122 Da) occurred. In the low-mass region, abundant ions m/z 311/313, 293/295, 189/191 corresponding to the fragmentation of a single 2,3-di-O-benzoyl-6-deoxy-6-bromo hexose unit along with m/z 105 (C6H5CO+) were observed.ESI MS was used for the characterization of regioselectively derivatized maltooligosaccharides obtained starting from natural β-cyclodextrin . For two2 inclusion complex was reported [2 + 2H]2+ and [C60:(γ-CD)2 + 2Na]2+, only [(γ-CD) + H]+ or [γ-CD + Na]+, respectively, were registered along with ionized fragments of γ-CD ([Glcn + H/Na]+), whereas for negatively charged ion [C60:(γ-CD)2 − 2H]2−, the major fragments gave superposed C60− and [C60 + H]− peak clusters along with minor superposed clusters of deprotonated cyclodextrin [γ-CD − H]−/[(γ-CD)2 − 2H]2−.An interesting observation concerning charge differentiation in tandem ESI CID mass spectra of C60:(γ-CD)reported . For posO-sulfated, polyethylene glycol α,ω-dimethylacrylate-tethered, dipyrenyl-terminated rotaxanes were profiled using Orbitrap ESI MS. Positive ion mode ESI MS2 was successfully applied for structure characterization of individual components in a mixture of rotaxanes.A mass spectrometric study of complex, supramolecular object was performed quite recently . PolydisO-isopropylidene and 3-O-dodecyl, were subjected to ESI MS2 fragmentation in triple quadrupole instrument using Ar as a collision gas. Schemes of low-energy CID fragmentation were proposed; the main losses were acetone (from isopropylidene) and dodecanol/dodecene (from dodecyl). Ether cycle cleavage also occurred; the authors included this fission as a central part in both schemes.A study of unusual, separately classified compounds was published in . These c2 fragmentation is now in progress; we believe that discoveries will happen concerning these unusual compounds in nearest future.For the last three decades, mass spectrometry of cyclic oligosaccharides has achieved great success, especially in the field of gas-phase chemistry of non-covalent, host-guest complexes. Nevertheless, some problems remain unsolved. First, there is no convenient and universal nomenclature of cleavages of cyclic oligosaccharides free of the reducing/nonreducing end formalism. No easy-to-use and reliable methods to differentiate between isomeric cyclic and acyclic oligosaccharides have been proposed. For induced decay of host-guest cyclodextrin complexes, no direct proof of guest-host proton transfer is presented. The boundaries of application of tandem mass spectrometry for determination of positions of substituents in cyclic oligosaccharides are not yet determined. A few works were done concerning fragmentation of negative ions of cyclic oligosaccharides. The methods of activation are now restricted by CID (ESI MS) and LID (MALDI MS), for example, no studies in ECD of multiply charged positive ions of cyclic CDs were carried out. A search of structure effects in complex, mixed cyclic oligosaccharides on their MS


Night shift work has become highly prevalent in our 24/7 societies, with up to 18% of the US work force working alternate shift schedules. However, studies indicate that there may be adverse health effects of chronic night work across diverse populations. These effects are likely due to misalignment of the circadian system with work schedules, mediated by the system’s primary marker melatonin as well as other downstream molecules.Melatonin has multiple biologic actions that are relevant to cardiometabolic disease, including modulation of oxidative stress, inflammation, and (via the melatonin receptor) vasoconstriction. Behavioral traits, such as chronotype and meal timing, have recently been shown to interact with the effects of night work on cardiometabolic health.Together with recent findings suggesting a role for circadian genes in cardiometabolic risk, the interactions of night shift work and behavioral traits are likely to facilitate novel treatment and prevention approaches for cardiovascular disease and type 2 diabetes, incorporating aspects of clock and timing. Involvement of the circadian system in the pathogenesis of cardiovascular disease (CVD) has been suspected since older anecdotal evidence indicated a higher frequency of myocardial infarction in the early morning hours. More recent reports have continued to support this notion . In the A number of epidemiologic studies have explored the association between shift work and diabetes in recent decades. The majority of these studies were conducted since 2000 across the United States (US), Europe, and Asia. A recent meta-analysis identified 12 such studies involving more than 200,000 participants, and reported that, overall, shift work history was related to an increased risk of diabetes . AlthougPtrend < 0.001). Specifically, compared to participants without such a history, participants with 1–2, 3–9, 10–19, and ≥ 20 years of rotating night shift work history had the following elevated risks of diabetes after adjustment for important confounding factors: hazard ratios (HRs) 1.05 (95% CI = 1.00–1.11), 1.20 (1.14–1.26), 1.40 (1.30–1.51), and 1.58 (1.43–1.74), respectively. These results were consistent across the two cohorts. Additional analyses adjusting for body mass index attenuated these estimates, although the overall trend remained highly significant; still, this result suggests that body weight might mediate the observed association.A landmark study of rotating night shift work and diabetes  was conducted by our group within the Nurses’ Health Study (NHS) and Nurses’ Health Study II (NHS II) in 2011 . This st•]. In particular, a mismatch in sleep and work timing appeared to increase the risk of diabetes slightly among early chronotypes who worked ≥ 10 years of rotating night shifts, and to increase the risk strongly among late chronotypes who worked no rotating night shifts. This finding is consistent with recent evidence suggesting a role of meal timing  have been identified in all nucleated cells –30 that The circadian system is controlled by the “master pacemaker,” which regulates the sleep/wake cycle and pineal melatonin biosynthesis . It alsoBehaviors that induce misalignment between internal clock and external timing  have the potential to upset the inherent, roughly regular circadian rhythm in humans. Several studies have demonstrated negative effects of circadian misalignment on measurable biomarkers, including melatonin. Levels of melatonin’s major urinary metabolite, 6-sulfatoxymelatonin (aMT6s), are closely correlated with nightly peak plasma melatonin levels in blood and saliva, and therefore allow determination of nightly peak levels of melatonin in human studies . In addi•]). Additional evidence for the protective effect of circulating melatonin on CVD/T2DM risk comes mostly from cross-sectional studies reporting decreased melatonin levels  in patients with versus without diabetes [Human observational studies have shown higher melatonin levels to be inversely associated with higher melatonin levels to be inversity associated with hypertesion, T2DM and incident myocardial infarction (MI). For example, in prospective, nested case-control studies of the NHS, after adjusting for an extensive set of confounding variables, the odds ratio for a unit decrease in log-transformed sulfatoxymelatonin:creatinine ratio was 1.48 (95% CI = 1.11–1.98) for T2DM and 1.40 (95% CI = 1.02–1.93) for incident MI •). Additdiabetes , 43, anddiabetes , 45.Several lines of evidence from experimental studies support that melatonin may have a beneficial effect on glucose metabolism  and cardMTNR1B (type 2 melatonin receptor) and T2DM, with odds ratios ranging from 1.09 to 1.20. Such findings provide further support for an association between the circadian system—and hence night shift work, which adversely affects this system—and cardiometabolic health [CRY2 gene has been associated with increased insulin resistance and T2DM at genome-wide levels of significance [Several associations between circadian gene variants and health outcomes have previously been described (e.g., sleep disorders  and obesc health –73. Addiificance .In summary, several lines of evidence, both from mechanistic studies as well as observational data in humans, provide support for a causal association between circadian disruption as encountered by night shift workers, and the primary circadian marker melatonin, with risk of T2DM and CVD. While night workers have been found to experience a higher risk of cardiometabolic diseases including T2DM and CVD, the mechanistic studies suggest that these effects are mediated by alterations in melatonin levels and melatonin’s function on melatonin receptors throughout the body, as well as through clock-genetic variants, and inflammatory and endothelial pathways common to CVD and T2DM.There is ample evidence for a variety of important pathways that are, at least partially, under clock control and suggest close involvement of the circadian system in cardiometabolic pathophysiology. Recent studies of behavioral traits, such as chronotype and corresponding meal timing, also suggest an interaction with night shift work , and fut


The pharmacokinetics of four upadacitinib extended-release formulations with different in vitro release characteristics and an immediate-release capsule formulation of upadacitinib were evaluated in 20 healthy subjects in a single-dose, randomized, crossover study. In vivo pharmacokinetic data and in vitro dissolution data  were used to establish a level A IVIVC. Three formulations were used to establish the IVIVC, and the fourth formulation was used for external validation. A non-linear IVIVC best described the relationship between upadacitinib in vitro dissolution and in vivo absorption profiles. The absolute percent prediction errors (%PE) for upadacitinib Cmax and AUC were less than 10% for all three formulations used to establish the IVIVC, as well as for the %PE for the external validation formulation and the overall mean internal validation. Model was cross-validated using the leave-one-out approach; all evaluated cross-validation runs met the regulatory acceptance criteria. A level A IVIVC was successfully developed and validated for upadacitinib ER formulation, which meets the FDA and EMA regulatory validation criteria and can be used as surrogate for in vivo bioequivalence.Upadacitinib is a selective Janus Kinase 1 inhibitor which is being developed for the treatment of several inflammatory diseases including rheumatoid arthritis. Upadacitinib was evaluated in Phase 3 studies as an oral extended-release (ER) formulation administered once daily. The purpose of this study was to develop a level A The online version of this article (10.1208/s12248-019-0378-y) contains supplementary material, which is available to authorized users. Upadacitinib ABT-494) is a novel, selective Janus kinase (JAK) 1 inhibitor that potently inhibits JAK 1, but is less potent against other JAK isoforms –4. Upada94 is a nCmax and Cmin to 6 mg and 12 mg BID using the IR formulation under fasting conditions [in vitro assessments, upadacitinib has been shown to be highly permeable and highly soluble at clinically relevant doses across the pH range of 1 to 7.5 (data on file at AbbVie). Based on in vitro assessments, upadacitinib is considered to be a class I drug according to the Biopharmaceutics Classification System [Upadacitinib pharmacokinetics was characterized in healthy subjects following the administration of the immediate-release (IR) and the extended-release (ER) formulations ,12. The n System . The relin vivo clinical study that is adequately powered to demonstrate bioequivalence [in vivo–in vitro correlation (IVIVC) can allow for the prediction of the plasma concentration time profile of a formulation without having to conduct in vivo bioavailability studies, thus saving time and costs and avoiding the need to administer a drug to healthy volunteers [in vivo bioavailability studies which may be needed to approve and maintain a drug product on the market [Throughout the life-cycle of a drug product, the need often arises to change some aspects of the formulation, the manufacturing process, or the manufacturing site. Some post-approval changes require the need to demonstrate bioequivalence between the modified and the marketed formulation through an ivalence ,18. Furtlunteers . The avae market ,18.in vitro dissolution and in vivo pharmacokinetics for four ER formulations (30 mg strength) with various in vitro release profiles, including the proposed commercial formulation, were evaluated relative to a 24-mg dose of the immediate-release (IR) capsule formulation. The methodology for the evaluation and establishment of a non-linear level A IVIVC for upadacitinib ER formulations are described.In the present study, the in vivo clinical study.Four ER formulations  were developed with identical amounts of upadacitinib (30 mg) but varying amounts of the release-controlling polymer HPMC. Three formulations  contained 10%, 15%, and 35% HPMC, respectively. The fourth formulation evaluated (Formulation C) contained 20% HPMC as a prototype for the planned commercial formulation. A single 24-mg dose of upadacitinib (2 × 12 mg IR upadacitinib capsules) was used as the reference formulation in the in vitro dissolution profiles. The dissolution medium was 900 mL of 0.05 M phosphate buffer (pH 6.8). An automatic sampler collected 1.5 mL of each of the sample at multiple time points , and the samples were then analyzed with high-performance liquid chromatography (HPLC). Several additional dissolution methods were also evaluated .A USP Dissolution Apparatus 1 operating at a rotating speed of 100 rpm was used to generate the N = 20) in general good health were selected to participate in the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee  and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individuals included in the study prior to any study-related procedure. Enrolled subjects were randomly assigned in equal numbers to one of five sequences of regimens A, B, C, D, and E consisting of five periods. In each of the five periods, a single dose of upadacitinib was taken orally with approximately 240 mL of water after a fasting for a minimum of 10 h and at least 4 h before lunch. Each dose was separated by a washout interval of 4 days. Blood samples for upadacitinib assay were collected into dipotassium ethylenediaminetetraacetic acid-containing collection tubes prior to dosing (0 h) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 72 h after dosing in each period. Plasma concentrations of upadacitinib were determined using a validated liquid chromatography method with tandem mass spectrometry [A Phase 1, single-dose, open-label study conducted according to a five-period, randomized, crossover design was used to characterize the bioavailability of four upadacitinib ER tablet formulations with different dissolution release rates relative to upadacitinib immediate-release capsules under fasting conditions. The study was conducted at PPD Development , and subjects were confined to the study site and supervised for approximately 21 consecutive days. Adult male subjects (trometry . The lowCmax) of upadacitinib and time to Cmax (tmax) values were determined directly from the plasma concentration versus time data for each subject. The apparent terminal phase elimination rate constant  was obtained from the slope of the least squares linear regression of the logarithms of the plasma concentration versus time data from the terminal log-linear phase of the profile. The terminal phase elimination half-life (t1/2) was calculated as ln (2)/β. The area under the plasma concentration versus time curve from zero to the last measurable concentration (AUCt) and area under the plasma concentration versus time curve from zero to infinity (AUCinf) were calculated by the linear trapezoidal method.Pharmacokinetic parameters were estimated using non-compartmental methods in Phoenix® Version 7.0 . The maximum observed plasma concentration , and b is the slope factor.in vitro dissolution data and the Akaike Information Criteria (AIC) from each equation [in vivo data. The individual UIR parameters were estimated by fitting a maximum of three polyexponential function with a time lag to each individual profile from the IR formulation and choosing the one which best fits the profile based on the AIC with a uniform weighting scheme. Since the reference formulation was an IR formulation, the “strip Ka” approach was followed to ensure that the UIR is decoupled from the absorption process as previously described [Tlag) was incorporated into the model to allow for a time lag in absorption. The adequacy of the selected model was determined through evaluating the agreement between model-predicted and observed upadacitinib plasma concentration versus time profiles.Individual unit impulse response (UIR) parameters were generated using the IR formulation escribed . This asin vivo (Fa) from each of the four upadacitinib ER formulations relative to the IR formulation was estimated by numerical deconvolution of the observed plasma concentration versus time profiles against the UIR from the reference IR formulation of upadacitinib. Exploratory plots were generated to evaluate the relationship between Fa and Fdiss, vitro as well as between the in vitro dissolution time (Tvitro) and in vivo absorption time (Tvivo).The fraction of upadacitinib dose absorbed Cmax and a linear IVIVC could not be established. A user-specified non-linear IVIVC was evaluated by fitting a non-linear Emax model to scale the Tvitro to the Tvivo, as shown in Eq. Fa and Fdiss, vitro.Several linear and non-linear models were evaluated for correlation between the fraction dissolved versus time and fraction absorbed versus time profiles of upadacitinib. Three formulations  were used to establish the IVIVC, and the fourth formulation (formulation B) was used for external validation. Default linear models in Phoenix IVIVC toolkit which employ absorption scale, time scale, and/or time shift for different dissolution conditions were initially evaluated. However, all the evaluated linear models demonstrated under-prediction for upadacitinib The equation to use was selected based on adequacy of fitting the equation .IndividuA1, A2, and B2 are constants characterizing the Emax relationship between Tvitro and Tvivo.Fa  corresponding to Fdiss, vitro at different time points for each formulation. The Fa, pred for each of the four ER formulations and the individual UIRs from the IR capsule formulation were used as the input function for convolution to generate individual predicted plasma concentration versus time profiles for the different ER formulations. The predicted pharmacokinetic parameters, Cmax, AUClast, and AUCinf were estimated using non-compartmental analysis within the Phoenix IVIVC toolkit.The developed IVIVC relationship was used to predict inf and Cmax for each ER formulation as:The prediction error, %PE, was calculated for AUCAdditionally, a cross-validation was conducted using the leave-one-out approach. The final IVIVC model was re-run using each of the four extended-release formulations  as an external validation formulation and the remaining three formulations for model building and internal validation. The %PE was calculated for internal and external validation formulations for each of the cross-validation runs.Cmax and AUCinf was required. In addition, the absolute % PE for each individual formulation was required not to exceed 15%. For external validation, the absolute %PE was required not to exceed 10% for Cmax and AUCinf.The predictive ability of the IVIVC was assessed for the numerical IVIVC through evaluating each method’s internal and external predictability per FDA and EMA guidance documents ,18. To ein vitro drug-release profiles for the four upadacitinib ER formulations using the selected method (USP App 1 at pH 6.8) are shown in Fig. in vitro dissolution data as it provided better fit compared to a Hill function  and ER tablets (30 mg) are described in Table Tmax of upadacitinib was 2.0, 2.5, 3.0, and 3.0 h for ER formulations A, B, C, and D, respectively, as compared to 1.0 h for the IR formulation. Upadacitinib terminal half-life was similar across the ER formulations and comparable to that of the IR formulation (approximately 10 to 13 h).The mean plasma concentration versus time profiles from the ent Fig. . The ratFa and Fdiss, vitro as well as between Tvivo and Tvitro are presented in Fig. Fa, obs and Fd, obs is linear  as well as for the external validation formulation (formulation B) are presented in Table inf and Cmax for the ER formulations . The %PE for each of the individual ER formulations A, C, and D were also well below 15% for both AUCinf and Cmax. In addition, the external %PE for ER formulation B was less than 10%  of 10 to 35%. This range encompasses the target to be marketed formulation (formulation C) which contains 20% HPMC. The established IVIVC meets both the internal and external predictability per the FDA and EMA criteria and can potentially be used as surrogate for in vivo study if there is a need for formulation change (within the design space of the IVIVC) or manufacturing process change. Additionally, the established IVIVC enables setting the release specifications based on clinical relevance using the predicted range of upadacitinib exposures for formulations that fall within the proposed specifications. These analyses highlight the importance of evaluating non-linear models when linear IVIVC relationships cannot be established.A predictive level A non-linear IVIVC was established for upadacitinib ER formulation using Cmax, (data not shown) suggesting slower dissolution in vitro than in vivo dissolution that was not adequately accounted for using a linear time scaling factor. This was also the case for various other in vitro test conditions evaluated to mimic the in vivo conditions . Given the high permeability and high solubility of upadacitinib, intestinal transporters are not expected to have clinically relevant role in upadacitinib disposition Tvitro and Tvivo appeared linear up to approximately 8 h in vivo. An approach that is alternative to the use of non-linear model would be to establish a linear IVIVC with in vivo cutoff time (tcutoff). However, use of tcutoff implies ignoring collected in vivo and in vitro data after the selected tcutoff. Additionally, Phoenix software that does not allow estimating the optimal tcutoff value and tcutoff will need to be selected as an arbitrary value based on observed data. Both of the aforementioned points can potentially introduce bias in the analysis when tcutoff is used. We opted to use a non-linear IVIVC approach rather than use of linear IVIVC with tcutoff to be able to use the totality of the data thus to avoid ignoring any in vitro or in vivo data and to allow the software to estimate the best-fitting parameters for the non-linear relationship based on the data.It is worth noting that the relationship between The IVIVC model was developed while a Phase 3 study which evaluated the safety and efficacy of upadacitinib doses of 15 mg and 30 mg QD using the ER formulation was ongoing. The upadacitinib ER formulation is considered proportionally similar between the 15 and 30 mg strengths, and upadacitinib plasma exposures are linear over a wide range of IR and ER doses ,15. Therin vitro test can potentially serve as a surrogate for bioavailability testing as well as a tool to screen formulations and set the dissolution and drug-release acceptance criteria.The robustness of the model was evaluated through cross-validation using the leave-one-out approach; all cross-validation runs meet the acceptance criteria . This assessment, although not a requirement per regulatory guidance documents, demonstrate robustness of the IVIVC and that it is not sensitive to data from a specific formulation. With a validated IVIVC, an A robust non-linear level A correlation that meets the FDA and EMA validation criteria for both internal and external predictability was established for upadacitinib ER formulation. This IVIVC can be used as surrogate for bioequivalence studies in case of future formulation changes that are covered by the IVIVC release rates tested. This correlation will enable the setting of clinically meaningful dissolution specifications based on acceptable differences in plasma concentrations corresponding to the upper and lower limit of the dissolution specifications.ESM 1(DOCX 14 kb)