Vitis vinifera L.) namely, Early Muscat, Radmilovac Muscat, Banat Muscat, Italia Muscat, and Muscat Hamburg, with the addition of pectolytic enzyme in two different concentrations or without it (control). A total of 58 volatile aroma compounds were detected by means of combined gas chromatographic-mass spectrometric (GC/MS) method. Ethyl esters of C8–C18 fatty acids (21) and terpene (16) compounds were considerably more abundant in all grape brandy samples compared to the other volatile compounds identified. Pectolytic enzyme, positively affected terpenes content in the brandy of all studied cultivars. The similarities between brandy samples produced from Muscat Hamburg (MH) and other Muscat cultivars may be attributed to the parentage of MH to those cultivars.Grape brandy, known as ‘Lozovača’, is one of the most produced alcoholic beverages in the Republic of Serbia. Muscat cultivars are highly priced in grape brandy manufacturing. Among the numerous factors, cultivar-specific characteristics have a significant influence on its quality and aroma profile. Pectolytic enzymes play a part in increasing intensity of the prefermentative aroma by hydrolysis of terpenic glycosides, from which the compounds that contribute to the aroma of brandy are released. In this study, grape brandy samples were produced from five Muscat table grapevine cultivars ( European Union legislation describes brandy as an alcoholic beverage which is produced from wine spirit, with or without wine distillate, distilled at less than 94.8%  is especially valued because of the specific aroma. Owing to the richness of aromatic compounds in Muscat cultivars, they are often appraised as models for the study of flavor compounds [Muscat Hamburg is an economically very important table grape cultivar and is often highly priced due to its enjoyable taste . ‘Lozovaompounds ,17. HoweThe objective of this study was to evaluate the composition of the aromatic complex of the grape brandy ‘Lozovača’ produced with the application of pectolytic enzyme from five Muscat table grapevine cultivars. Domestic cultivars, Radmilovac Muscat and Banat Muscat, and the introduced cultivars, Early Muscat, Italia Muscat, and Muscat Hamburg, were used for the brandy production. The effect of the enzyme on the release of the aromatic compounds, as well as the similarities between the composition of brandy samples produced from Muscat Hamburg and other Muscat cultivars were discussed. In the individual samples of Early Muscat (EM), Radmilovac Muscat (RM), Banat Muscat (BM), Italia Muscat (IM) and Muscat Hamburg (MH) a total of 37, 35, 25, 32, and 27 aromatic compounds, respectively were identified that belong to different groups including acetals, alcohols, acids, esters, terpenes, ketones, and amides. The major aroma contributing compounds alcohols, acids, esters, terpenes are presented in Alcohols—All brandy samples  were found to contain 1-hexanol and phenyl–ethyl alcohol. Phenyl–ethyl alcohol was identified in all distillate samples except for the V2 variant in the brandy of the Radmilovac Muscat cultivar. The smallest percentage was recorded in the control sample of the Radmilovac Muscat distillate (0.42%) and the highest relative representation was recorded in the Muscat Hamburg V1 distillate (25.43%). The smallest content of phenyl–ethyl alcohol was recorded in the brandy of Italia Muscat cultivar  in comparison to other distillates. It is important to note that with the use of pectolytic enzymes in the Italia Muscat brandy, the relative content of phenyl–ethyl alcohol decreased. Notably higher relative content of 1-hexanol in relation to other cultivars were found in distillates from Banat Muscat , Radmilovac Muscat  and Muscat Hamburg , Acids—Short-chain fatty acids have not been identified in any sample (either in the control sample or in the variants with the implementation of pectolytic enzymes). Of the middle-chain fatty acids, octanoic, decanoic, dodecanoic and hexadecanoic acids were identified in most brandy samples  was observed between the samples of Muscat Hamburg, Banat Muscat and Italia Muscat. On the other hand, the effect of pectolytic enzyme on the relative abundance of terpenes in Early Muscat, Ramilovac Muscat and Banat Muscat samples had a similar pattern. Namely, in V1 samples (with the lower amount of enzyme applied) the higher relative content of terpenes was observed compared to the control sample and V2 samples, c-linalool oxide and, esters, ethyl hexanoate, ethyl decanoate, and ethyl dodecanoate  and hotrienol (RMV1). Close to RMV1 was EMV1, characterized by citronellol and linalool, while EMV2, in the bottom of the right quadrant, was separated from their counterparts by linalool oxides, indicating significant effects of different concentrations of used pectolytic enzyme on aroma profiles of EM brandies. A similar effect was observed for Muscat Hamburg; MHC and MHV2 were positioned in the bottom left quadrant due to high content of ethyl hexanoate, while MHV1 was more associated with higher content of limonene and ethyl tetradecanoate. The group of IM samples were positioned closely in the left bottom quadrant and were characterized by higher content of ethyl octanoate, decanoate and dodecanoate. Similarly, the group of BM samples located in the top right quadrant was separated by ethyl hexadecanoate (BMV1) and ethyl-9-hexadecanoate (BMV2). c-linalool oxide and hotrienol on PC1, and ethyl decanoate, ethyl-9-hexadecanoate and stearate on PC2. Separation of EM samples was mostly based on ethyl octanoate, dodecanoate, stearate and terpenoids, limonene, linalool, hotrienol and citronellol (PC1) and phenyl–ethyl alcohol, ethyl-9-hexadecanoate and t-linalool oxide (PC2). To explore the influence of used pectolytic enzyme on aroma profiles in more detail, principal component analysis was performed for each variety . While 2The agglomerative hierarchical clustering (AHC) of brandy samples without the addition of pectolytic enzyme grouped samples by their dissimilarity into three clusters; a cluster (C) with EMC, IMC and HMC, and two clusters that contained only RMC or BMC . The bigThe agglomerative hierarchical clustering (AHC) of brandy samples with and without the addition of pectolytic enzyme grouped samples into three clusters. The C1 included EMC, IMV1, IMV2, MHC, and MHV2, the second cluster (C2) contained EMV1, EMV2, RMC, RMV1, and RMV2 while the third cluster (C3) had BMC, BMV1, BMV2, IMC, and MHV1 . C1 was characterized by high content of ethyl esters: hexanoate, octanoate, decanoate, and low level of ethyl hexadecanoate and linoleate compared to the other two clusters. C2 was found to be quite opposite, the same esters were quite low but all terpenes high. C3 had a high content of phenyl–ethyl alcohol, ethyl hexadecanoate and linoleate, but low levels of terpenes: linalool, citronellol, and geraniol .v/v) that causes a burning sensation in the mouth and increases the sweetness. Grape brandies have a composite aroma profile with hundreds of volatile compounds found in grapes (primary aromas) and/or during wine fermentation (fermentation aromas), whereas some arise from the distillation process or through extraction from oak wood. Many significant aroma compounds are formed during grape ripening (monoterpenes) and during alcoholic fermentation . The organoleptic characteristics could be distinctive even though the volatile composition is fairly similar, and this is possibly because of slightly different concentrations of volatile compounds [Being a product of wine distillation, brandy does not contain the non-distillable wine organic and inorganic compounds. Non-volatile organic acids are not present in the distillate which affects the taste balance and the high content of alcohol in brandy , by enzymatic degradation and subsequent reduction of long-chain fatty acids [ty acids ,19. A sity acids . Accordity acids . In thisAcids—Short-chain fatty acids were not identified in any sample, either in the control sample or in any other variant with the application of pectolytic enzyme , overripe pomace and wines of Muscat cultivars are terpenes, linalool, geraniol and nerol, but also terpineol [α-terpinolene, hotrienol, rose oxide, citral, citronellol, manoyl oxide, myrcene, α-terpinene, and p-cymene, significantly contribute to the grape brandy aroma and are specific only for distillates obtained from grapes (Vitis vinifera L.) [erpineol . Muscat erpineol . Linalooerpineol . Terpeneerpineol . The suberpineol . Geranioerpineol ,37. The fera L.) ,16. c-linalool oxide, t-linalool oxide, limonene, and geraniol were the most abundant, and among them, the most dominant were linalool and citronellol. Other terpene compounds were detected sporadically and in significantly less relative content . Linalool was detected in all samples (1.73%–19.22%), whereas nerol was not present at all. The results obtained for the linalool content indicated its increase in the samples with added pectolytic enzyme of all studied cultivars except for Banat Muscat , except for BMC and BMV1. t-Linalool oxide was not identified only in Banat Muscat brandy samples, while in other samples its relative content ranged from 0.23%–3.37%. Limonene, associated with lemon and fruity aromas, was detected in the samples where pectolytic enzyme was applied , which may be attributed to the influence of the enzyme. The effect of compounds on the impartment of both odor and aroma is mostly induced by its abundance i.e., content. However, this is not the case with terpenic compounds and some esters which were noted for their low olfactive threshold values. Namely, the low detection threshold level indicates a high degree of contribution to the distillate aroma regardless of their low concentration. Linalool and geraniol, for example, with low detection threshold, have a far stronger aromatic character as compared to nerol that reaches identical odor intensity at four-fold concentrations [A total of 16 terpene compounds were identified and observed in all samples of the studied cultivars (control and with the application of enzymes). Among them, linalool, citronellol, hotrienol, t Muscat . Geraniot Muscat . Hotrient Muscat . c-Linalamples 0.%–3.21%, trations ,40,41. The most notable changes in relative abundance (%) were observed for esters and terpenes. The application of pectolytic enzyme influenced the increase of the relative content of esters in the samples of RM and MH. On the other hand, in brandy samples obtained from other studied cultivars, relative content of esters declined with the application of the pectolytic enzyme. Pectolytic enzyme, in both concentrations, positively affected terpene content in brandy samples of all studied cultivars. Riparia × Berlandieri Kober 5BB. Plant density is 3 m between rows and the distance between the vine-wood is 1 m. Plant population per hectare is 3333 vine-wood. For the production of the brandy, grapes of table cultivars Early Muscat (EM), Radmilovac Muscat (RM), Banat Muscat (BM), Italia Muscat (IM), and Muscat Hamburg (MH) were used. RM and BM are native cultivars bred in 1980s at the Faculty of Agriculture, whereas EM, MH and IM are introduced cultivars. Their description is given according to literature [Experiment was carried out on the plantation of grapevine cultivars at the Radmilovac Experimental Field . The geographical position of the plantation is at 44° 45′ N/20° 35′ E and 135 m above sea level on a mild slope exposed to the south. By its location, the plantation belongs to Belgrade area and Grocka vineyards. Grocka vineyards are characterized by favorable climatic conditions (vicinity of the Danube River) for the production of high-quality table grapes. The examined cultivars were inoculated on the vine rootstock terature ,43.o 304 -3- early). It was created by crossing the cultivars Muscat Hamburg x Queen of the Vineyard. Its cluster is of medium size, nice looking. The berries are oval, medium-sized, yellow, and white. The flesh is tendinous with a fine Muscat taste.Early Muscat is a table cultivar of early stage of ripening . The cultivars Queen of the Vineyard x Muscat Hamburg were used as parental partners. Its cluster is medium to large-sized, loose. The berries are large, round, the skin is yellow-green. The flesh is crispy with a pleasant Muscat taste.Radmilovac Muscat is an early table cultivar , created by crossing the cultivars Queen of the Vineyard x Muscat Hamburg. It has a medium-sized cluster. The berries are medium-sized, oval with dark reddish blue skin. The flesh is medium firm with a delicate Muscat aroma.Banat Muscat is an early table cultivar , created by crossing the cultivars Bicane x Muscat Hamburg. Its cluster is medium-sized or large, conical and loose. The berries are large, egg-shaped. The skin is thick, yellow-greenish or green-yellowish. The flesh is medium firm with a very pleasant muscat aroma.Italia Muscat is a table cultivar of late ripening period . Its cluster is medium to large-sized, loose and branched. The skin is solid with dark blue color. The berries are oval, uneven in size with a fine Muscat aroma.Muscat Hamburg is a cultivar of late ripening period .Grapes of each cultivar were harvested at the stage of its physiological full maturity, according to the method of O.I.V. . For makBrandy samples obtained from each cultivar were labelled as: C—control (without the addition of the enzyme), V1 (0.3 g of pectolytic enzyme per kg of grape) and V2 (0.7 g of pectolytic enzyme per kg of grape). For all samples analyzed, liquid–liquid solvent extraction with pentane was applied (100 mL brandy and 10 mL pentane) for each sample. Analysis of volatile compounds was performed by gas chromatography-mass spectrometry (GC/MS) method as previously described . BrieflyGas chromatographic-mass spectrometric (GC/MS) analysis was performed using an Agilent 6890 gas chromatograph coupled with an Agilent 5973 Network mass selective detector (MSD), in positive ion electron impact (EI) mode. The separation was achieved using an Agilent 19091S-433 HP-5MS fused silica capillary column, 30 m × 0.25 mm i.d., 0.25 μm film thickness. GC oven temperature was programmed from 60 to 285 °C at a rate of 4.3 °C/min. Helium was used as carrier gas, inlet pressure was 25 kPa, linear velocity was 1 mL/min at 210 °C. Injector temperature was 250 °C, and injection mode splitless. MS scan conditions: source temperature, 200 °C; interface temperature, 250 °C; E energy, 70 eV; mass scan range, 40–350 amu (atomic mass units). Identification of compound was performed using both the retention index and comparison with reference spectra (Wiley database). The relative percentage of the compounds identified was computed from the GC peak area. The principal component analysis (PCA) was used to study the similarity between Muscat cultivars, as well as the similarity of the measured parameters. This method is very useful when it is necessary to extract relevant information from complex datasets. This information was obtained by reducing the dimensionality of the space in which the measured data are scattered. The Principal components are linear combinations of the initial variables  and thus the first main component is selected to absorb as much variance of the original data as possible, the second as much as possible from the remaining variance, etc. All Principal components were mutually orthogonal. The visualization of the PCA was achieved by the construction of the biplot, on which the cultivars were represented by points and the oenological parameters to the vectors. Agglomerative hierarchical clustering (AHC) was conducted in Addinsoft 2018.5 XLSTAT Sensory, MS Excel, Addinsoft NY USA. The results obtained in this study on volatile aromatic compounds in the analyzed grape brandies suggest significant differences in both the number of aromatic compounds and their relative content. Given the uniform grape brandy making technology, the differences observed were induced by the cultivars used for grape brandy production and possibly the length of their ripening period. The application of pectolytic enzyme did not influence evenly the increase of relative percentage of aromatic compounds in the examined brandy samples. Esters and terpenes were detected in the highest percentage, which may be attributed mainly to fruity and floral aromas of brandy samples. However, among terpenes, the main aroma contributors, the presence of limonene was associated only with the application of pectolytic enzyme. Principal Component Analysis revealed that esters and terpenes had the highest influence on discrimination between the Muscat cultivars. The Hierarchical Component Analysis of brandy samples without the addition of pectolytic enzyme (control) denotes a significant similarity among the cultivars, particularly between Muscat Hamburg and Italia Muscat, which may be attributed to the fact that Muscat Hamburg is one of the parents to the other examined cultivars. The initial examination of the applied enzyme effects imposes the need for further experiments and the use of other types of enzymes in order to compare their effectiveness in releasing aromatic compounds and improving brandy aroma.


Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo. Histone post-translational modifications play a major role in regulating access to DNA, and as such are important in the regulation of gene transcription and DNA repair. Due to this biological role, a significant amount of research focuses on creating a better understanding of the proteins that regulate histone modifications. One such modification, histone acetylation, is regulated by lysine acetyltransferases ) and histone deacetyltransferase (HDACs). One of the most studied KATs, p300, is a prolific acetyltransferase, with mutations in p300 resulting in several diseases including neurological disorders and cancer ,2,3,4,5.There are two major obstacles to better understanding the role of histone acetylation in maintaining a healthy cellular state. The first is the sheer number of potential sites of acetylation on histones: Nucleosomes are made up of an octamer, consisting of two copies each of histone H2A, H2B, H3, and H4. On this octamer there are dozens of lysine residues that have been shown to be targeted for acetylation in a biological context. Additionally, each KAT demonstrates specificity for particular lysine residues, emphasizing the importance of understanding the mechanisms underlying site-specific histone modification. This first obstacle can be addressed by utilizing mass spectrometry analysis, which allows the simultaneously quantitation of modification to multiple lysine residues ,7.N-[4-chloro-3-(trifluoromethyl)phenyl]-2-ethoxy-6-pentadecylbenzamide) was classified as the first known small molecule activator of p300 [N-(4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide) is a simplified analogue of CTPB [The second major obstacle to investigations into the biological impact of histone acetylation is the quality of currently available small-molecule regulators of histone acetyltransferases, and p300 in particular. For this investigation, we studied several putative p300 targeting compounds, including EGCG, CTPB, CTB, and garcinol (described below), while also continuing our characterization of C646, which we have previously studied . Epigall of p300 , while C of CTPB . CTB is  of CTPB . Finally of CTPB ,17. For these reasons, many of these small molecules are not ideal compounds for the modulation of histone acetylation. Indeed, recent studies have been undertaken to further pinpoint the off-target effects of reported histone acetyltransferase-targeting drugs compounds . DespiteThus, this work can serve as a template for studying the effects of potential histone acetylation-altering compounds, help to identify potential issues with these compounds, and provide valuable information on their in vitro and in vivo activity. Importantly, the methodology we have developed can be utilized to help address the lack of highly specific KAT-targeting compounds by providing a rapid and efficient means for identifying and testing novel p300-targeting drugs in the future.Reagents—All Chemicals were purchased from either Sigma-Aldrich  or Fisher Scientific ; the purity was the highest commercial grade available or met LC/MS grade. Ultrapure water was generated from a Millipore Direct-Q 5 ultrapure water system . Recombinant histone H3 and H4 were purified and provided from the Protein Purification Core at Colorado State University. Acetyl-CoA was obtained from Sigma-Aldrich. Garcinol and CTPB were purchased from Abcam , CTB from Sigma-Aldrich, EGCG from Cayman Chemical, and C646 from EMD Millipore.Protein purification—Full length p300 was expressed and purified from Sf9 cells as previously described [escribed . The p30Kinetic experiments—Experiments to characterize the effect of various drugs on p300 acetylation were conducted under kcat conditions in buffer containing 100 mM Ammonium bicarbonate and 50 mM HEPES buffer (pH 7.8) at 37 °C. All reactions were performed in the presence of 0.2% DMSO. Experiments were performed utilizing excess H3/H4 (15 µM) and acetyl-CoA (200 µM) in the presence of p300 (5 nM). A time course was performed to determine the rate (v/E) of acetylation. Assays were quenched using four volumes of trichloroacetic acid (TCA), and prepared for mass spectrometry analysis as previously described [etyl-CoA 0 µM in tMeasurement of acetylation from cell culture samples—Histones were extracted overnight from cell pellets using 0.2 N HCl. Histones were precipitated with trichloroacetic acid (TCA), and prepared for mass spectrometry analysis as previously described [escribed . QuantifTreatment of zebrafish embryos—Embryos were treated 0.5 h post fertilization with either 0.2% dimethylsulfoxide (DMSO) or the indicated dose of C646, EGCG, CTB, CTPB, or garcinol. Groups of 110–130 embryos were arrayed into six-well plates with a total of 1.5 mL of E3 zebrafish embryo media. For histone extraction, embryos were washed with ½ x Ringer’s solution, before being lysed with Triton extraction buffer (PBS containing 0.5% Triton X 100 (v/v), 0.02% (w/v) NaN3). Histones were extracted overnight using 0.2 N HCl. UPLC-MS/MS analysis—A Waters Acquity H-class UPLC  coupled to a Thermo TSQ Quantum Access  triple quadrupole (QqQ) mass spectrometer was used to quantify acetylated H3/H4 peptides as previously described [escribed ,21.QqQ MS data analysis—Each acetylated and/or propionylated peak was identified by retention time and specific transitions, as previously reported [pF) is calculated by equation 1, where sI is the intensity of a specific peptide state and pI is the intensity of any state of that peptide, and analyzed as previously described [reported ,21. The escribed .(1)FP =Data analysis—All models were fit to the data-using Prism . The initial rates (v) of acetylation were calculated from the linear increase in acetylation as a function of time prior to 10% of the sum of acetylated residues. For comparisons of v/E  or fraction acetylation (in cellular and zebrafish samples), the significance of the change in rate was calculated using Prism, via two-tailed, unpaired t-tests. In vitro drug titrations were fit using a single-phase inhibition or a two phase model where the first is activating and the second is inhibiting . Computational docking and analysis—The structure of p300 1 was used as a template for docking experiments. Using PyMol2 , the bisubstrate analogue inhibitor bound in this structure, Lys-CoA, was split into acetyl-CoA and lysine portions for later binding site analysis. For docking, all bound ligands, including the five bromine atoms and all waters, were removed from the structure. The prepare_receptor4.py script included in AutoDock Tools3  was used to merge non-polar hydrogens, add Gasteiger charges, and assign AutoDock4 atom types to prepare the protein for docking. Using ChemDraw v14.0  followed by the OpenBabel4 babel program, the five ligand molecules  were prepared as energy minimized 3D models using the MMFF94 force field. 5 ) on the Wesleyan University High-Performance Computing Cluster. A shell script was written to submit 20 jobs generating 20 conformations each in parallel, for a total of 400 poses for each of the five ligands. The exhaustiveness parameter was set to 50, 2 CPUs were used for each job, with all protein residues kept rigid, but all single bonds in the ligands freely rotatable.Using the AutoDock Vina plugin for PyMol*, a grid box was designed to include the bound Lys-CoA (taken as an approximation of the location of the acetyl-CoA and lysine substrates) and all protein atoms within 5 Å. The  dimensions of this box were  for the center and  for the size. A second grid box was designed to include the entire protein. The  dimensions of this second box were  for the center and  for the size. Docking was performed using AutoDock VinaAfter docking, the resulting poses were analyzed to obtain the number and identity of the close contacts between each ligand and the protein residues, as well as the binding data from each pose. By examining the ligand interactions with residues within 5 Å of either the acetyl-CoA or the lysine parts of lys-CoA analogue, each ligand was assigned to one or more binding site. These data were organized in R and graphed using the ggplot2 package, and the 3-dimensional structures of the results were visualized using PyMol.We defined regions of the acetyl-CoA pocket on the basis of the binding regions of a lys-CoA molecule. Residues for the Lysine pocket: ARG1627, ASP1444, CYS1438, ILE1395, SER1396, TRP1436, TYR1397, TYR1446; residues for the acetyl-CoA pocket: ALA1437, ARG1410, ARG1462, ASP1399, CYS1438, GLN1455, HIS1451, ILE1395, ILE1435, ILE1457, LEU1398, LEU1463, LYS1407, LYS1456, LYS1459, PRO1439, PRO1440, PRO1458, SER1396, SER1400, THR1411, TRP1436, TRP1466, TYR1397, TYR1414, TYR1446, TYR1467). The non-active site pockets were identified by examining all of the consensus residues within 5 Å of a given ligand, when bound away from the active site. The residues for each of the two pockets are: pocket 1 - SER1329, ASP1330, LYS1331, THR1332, PRO1354, TYR1355, ARG1356, THR1357, LYS1358, GLU1380, TYR1381, GLY1382, SER1383, ASP1384, CYS1385, LEU1428, TYR1430, PRO1611, ILE1612, VAL1613 ASP1614, PRO1615, ASP1616, PRO1617, LEU1618; and pocket 2 - GLU1348, MET1349, ALA1350, PHE1353, PRO1386, PRO1387, PRO1388, ASN1389, GLN1390, ARG1391, ARG1392, THR1431, THR1432, GLY1433, HIS1434, ASP1482, TYR1483, LYS1484, ILE1486, GLN1489, ALA1490, GLU1492, ASP1493, ARG1494, LEU1495, SER1497, LYS1499, GLU1500, LEU1501, PRO1502, VAL1597, ILE1598, ARG1599, LEU1600, ILE1601, ALA1602, GLY1603, PRO1604, ALA1606, ASN1607, SER1608, LEU1609.A fundamental understanding of how a compound affects p300 comes from in vitro assays. In order to quantify site-specific changes in p300 histone acetylation activity, we utilized selected reaction monitoring (SRM), a quantitative MS-based approach that enables the simultaneous measurement of acetylation of multiple lysine residues ,20,23,24cat) of an acetyltransferase changes in response to external factors, such as drug treatment, can be utilized as a predictor of how that factor will alter the selectivity of a KAT in cells [cat of p300, time-course assays were performed under steady-state conditions in the presence and absence of our selected drugs. Under steady-state conditions, p300 activity is concentrated at the H3 and H4 tails residues [cat values for p300 at these residues in 0.2% DMSO has been previously determined [To characterize p300 histone acetylation activity, we began by measuring the catalytic efficiency of p300 at multiple sites of histone H3/H4; we have previously shown that determining how the site-specific catalytic efficiency  cannot be tested in vitro.For these experiments, we performed a titration of each drug in BxPC3 cells (human pancreatic cancer), which we used previously to test C646, as BxPC3 cells have no reported mutations in p300 nor CBP . We measThese results provide an important picture of the discrepancies that can be seen when screening the effect of KAT targeting drugs in vitro versus in cells, highlighting the importance of testing compounds in various systems, and providing a better biological context to the actual efficacy of such drugs. Thus, to further investigate the response to these drugs in a biological context, we continued our investigation using a zebrafish model system. We next tested how treatment with these drugs affects the biology of a healthy organism. While it is possible that drug treatments will alter the biology of cells in culture, changes were not obvious and would require more extensive molecular analysis. To further expand on our in vitro and cellular findings, we tested for survival and phenotypic changes in zebrafish embryos . These eEmbryos were exposed to drug at the 1-cell stage and survival was scored at 6 and 24 h post-fertilization (hpf). Groups of embryos contained 110–130 embryos arrayed on a 6 well plate. Phenotypes listed in We found that the embryos treated with up to 25 µM C646 survived up to 24 hpf, while causing death at higher concentrations. Treatment with garcinol and CTB at 25 µM and 10 µM respectively, resulted in death earlier, at 6 hpf. At 24 hpf, 10 µM of garcinol was also causing widespread embryonic lethality, although 1 µM CTB and Garcinol was well tolerated at 24 h (data not shown). Meanwhile, nearly 100% of the embryos survived treatments with CTPB and EGCG. As the intention of collecting this data was to make connections between survival and changes in histone acetylation, we noted that decreases in H3K9 acetylation levels observed in cells correlated closely with embryo death (see Discussion). To determine the feasibility of directly measuring histone acetylation changes in the embryos, we focused on C646 treatment, performing acid extraction of histones from pools of control- or C646-treated embryos at 24 hpf. These samples were also analyzed using our mass spectrometry-based method G–J. ConsIn silico modeling is a popular form of drug screening, with several p300 targeting compounds discovered via this type of analysis. As such, we were interested in determining whether the observed effects of p300 targeting drugs correlated in anyway with where and how they bind to p300. Thus, we performed in silico docking experiments to determine compound interaction with the catalytic portion of the KAT domain. Though the KAT domain of p300 represents only 16% of the overall structure, it is catalytically active in the absence of other domains ,29. To sWhen constraining the docking to the active site, all of the ligands bound in a competitive fashion with either histone, acetyl-CoA, or both. To quantitate this observation, the tunnel that defines the active site was divided into two different regions: the histone lysine pocket and the acetyl-CoA pocket. We defined regions of the acetyl-CoA pocket on the basis of the binding regions of a lys-CoA molecule (see Methods) A. CarefuWhen each of these compounds was docked to the entire KAT domain of p300 we observed that all of the C646 poses bound in the active site, while greater than 45% of the ligand poses for Garcinol and EGCG were in pockets on the opposite face from the active site (see Methods for residue numbers) . While iWhile these in silico binding experiments are a valuable tool for identifying compounds that bind to p300, data obtained from docking to the active site (either the binding position or variation in affinity frequency) failed to accurately predict in vitro or in vivo (in cells) specificity changes in the presence of drugs. However, the docking to the entire KAT domain of p300 revealed that selective in silico active site binding was associated with in vivo inhibition of p300. cat analysis in vitro, as an initial indication of the efficacy and site-specific effects of the drugs on histone acetylation. Subsequent experiments in cells allow us to further characterize these compounds in a more complex biological environment, allowing us to identify discrepancies between in vitro and in vivo behavior. The use of a zebrafish model also allows us to determine tolerable dosage ranges for these drugs and potential deleterious effects of the screened compounds. Utilizing increasingly complex models ensures the most efficient and effective use of resources, while providing valuable information about the drugs at each stage. By identifying relevant compounds and providing a steady stream of data to create meaningful connections between kinetics, cellular data, and in vivo treatment, this methodology provides a means for continuous discovery and characterization of novel molecular biology tools. A collection of highly specific and effective regulators of lysine acetyltransferases are currently lacking, limiting our ability to accurately probe the effects of histone modification independent of other off-target effects. Contributing to this problem are the unique challenges presented by multi-targeting enzymes, which hinder rapid and accurate identification of the compounds affecting them. Here we have investigated the efficacy of several previously identified p300-targeting compounds, while using this investigation as a mechanism for outlining a combined methodology for a rapid screen for classifying KAT-targeting compounds. This method begins with a kAs we would predict, we have found that drugs that are more specifically targeted to KATs have a closer relationship between their in vitro characterization and the changes in acetylation they bring about in cells. For example, we have seen in the past that the changes seen with C646 in vitro closely mimic the trends seen in cell culture . This coRecently, significant effort has been put into developing compounds that are more specific for p300 (and CBP). For example, a publication by Lasko et al. outlines a methodology for developing a highly specific p300 and CBP inhibitor. In this investigation, the authors also utilize a multifaceted approach in order to design and test a specific inhibitor: Their process starts with a computer-based virtual ligand screening, followed by in vitro and in vivo testing. Doing so, they are able to generate a compound that is highly specific to p300 and CBP. This type of investigation highlights the importance of the type of methodology we outline here, one which utilizes multiple systems to test and classify purported acetyltransferase targeting compounds. This type of repeated and varied testing, as we suggest here, is key to avoiding false hits that may appear in either in vitro, in silico, or even in vivo experiments when performed alone.In addition to outlining an efficient screen for KAT-targeting compounds, our results emphasize the importance of understanding changes in selectivity of multi-targeting enzymes. We see, both in vitro and in vivo, that the tested compounds consistently alter histone acetylation in a site-dependent manner , and neiAlthough the specificity of the tested compounds has largely proven to be subpar, they may have provided us with an interesting investigative lead that deserve further attention: Comparing our cell data with our zebrafish data, we noted that decreases in H3K9 acetylation levels observed in the cellular work  correlatFinally, of note, using the KAT domain of p300, we have shown that the in silico binding affinity can broadly predict whether p300 inhibition will correlate with in vitro and in vivo results–whereby the compounds with the highest affinity and that target the active site selectively (when docked against the entire protein) will correlate most strongly. Additionally, this study showed multiple different binding modes of our screened drugs, and identified distinct regions within the acetyl-CoA binding pocket for these small molecules to bind . We haveIn summary, we have demonstrated discrepancies in the in vitro, in vivo and in silico methods for predicting efficacy of several putative p300-targeting compounds. We have worked to generate a multi-faceted approach to more accurately identified the biological impact of compounds that target lysine acetyltransferases. We have shown an efficient way to identify and characterize the site-specific nature of these drugs, as well as a means for testing the potency and tolerable levels of these drugs. This methodology will be invaluable for developing more precise molecular biology tools to utilize for the study and treatment of diseases that are associated with aberrant histone acetylation.


Although current models of care are generally well-suited to providing treatment for individual medical conditions, the emergence of multimorbidity is becoming a serious concern for practitioners and policy researchers, particularly in developing countries. The challenges of tackling multimorbidity are further compounded when the multimorbidity co-occurs with psychiatric conditions such as cognitive and depressive disorders. Understanding the relationships between multimorbidity and psychiatric illnesses is therefore of considerable clinical importance. In the present study, we cross-sectionally examined whether multimorbidity has an association with perceived cognition—including memory, learning complaints, and depression—among elderly population in South Africa. Study subjects were 422 men and women aged 50 years and older. The prevalence of arthritis, asthma, cancer, diabetes, heart disease, chronic lung disease, hypertension, and stroke was respectively 31.5, 7.3, 1.7, 10.2, 1.2, 1.7, 52.1, and 31.5%, and that of multimorbidity was 30.8%. In the multivariate analysis, women with multimorbidity were 4.33 times  more likely to report memory complaints. The odds of diagnosed depression were 1.4 times , and the odds of self-reported depression were 1.7 times  higher among women who had multimorbidity compared with those who had no morbid conditions. However, the association was not significant among men. Overall, the findings suggest that the occurrence of multimorbidity warrants special attention, especially regarding its compounding effects on psychological health. The findings need to be replicated through longitudinal studies that consider a broader range of chronic conditions. Multimorbidity, in general terms referring to the coexistence of more than one chronic condition in the same person, is becoming a major concern for healthcare systems, especially in resource-poor countries. This is largely because primary care settings are typically configured to providing services for individual disease conditions rather than multiple diseases presenting in the same patient . As a poMDD and mild or brief depressive episodes are regularly encountered in primary care settings among the elderly, and are considered a global health issue. However, definitive evidence on the causal relationship between ageing and depression is still lacking. Less controversially, old age can exert negative effects on mental health outcomes through the occurrence of physical morbidity/co-morbidity and associated disabilities. However, the current literature regarding the relationship between multimorbidity and mental illness is inconclusive, particularly in countries in Sub-Saharan Africa. For this reason, an attempt was made in the present study to examine whether or not occurrences of single or multiple morbidity are associated with depression in the elderly population in South Africa. Because of the scarcity of relevant primary data, we used secondary data from the South Africa—SAGE Well-Being of Older People Study (WOPS) that had been conducted in 2010.This study was based on secondary data obtained from South Africa—SAGE Well-Being of Older People Study-2010 (WOPS) Wave 1. The WOPS cross-sectional survey was designed by the World Health Organization and the Africa Centre for Health and Population Studies and implemented by the Africa Centre for Health and Population Studies .Difficulty experienced last 30 days, concentrating/remembering thingsDifficulty experienced last 30 days, learning a new taskAnd, Ever diagnosed with depressionFelt sad, empty, or depressed last 12 monthsThe principal outcome variables were classified into two sub-domains, including cognitive complaints and depression, each of which were evaluated by two self-reported questions: To assess cognitive complaints, participants were asked: (1) “Overall, in the last 30 days, how much difficulty did you have with concentrating or remembering things? ;” and (2) “Overall, in the last 30 days, how much difficulty did you have in learning a new task ?” Subjects were given the following choices of response: none/mild/moderate/severe/extreme/cannot do.To assess depression, participants were asked: (1) “Have you ever been diagnosed with depression?” and (2) “During the last 12 months, have you had a period lasting several days when you felt sad, empty, or depressed?” Here subjects were asked to respond ‘yes’ or ‘no’.The principal explanatory variable was the frequency of the occurrence (self-reported) of following chronic diseases: arthritis, asthma, cancer, diabetes, heart disease, chronic lung disease, hypertension, stroke. Frequencies were categorized as: no morbidity (0 conditions), single morbidity (1 condition), and multimorbidity (>1 condition).To account for possible confounding effects, the following covariates were considered: age , gender , marital status , educational attainment , financial situation compared to 3 years ago , current tobacco use , average times had an alcoholic drink during last 12 months , Body Mass Index , self-reported health .Basic characteristics of the study population were presented as crude percentages. All estimations were stratified by gender. A contingency table was generated to facilitate the comparison of the prevalence rates of cognitive complaints and depression across sociodemographic variables. Chi-square bivariate tests of association were used to calculate the p-values. Frequency of diseases was trichotomised as ‘none’, ‘1’, and ‘>1’. The categories of memory and learning complaints were collapsed into- ‘none’, ‘mild/moderate’, and ‘severe/extreme’. Multivariate regression models were then used to calculate the odds ratios of the association (from adjusted models) between the outcome variables with the frequency of diseases by treating those with no diseases as the reference category. A two-tailed p-value of <0.05 was set as level of significance for all calculations. All analyses were carried out using R statistical software.The WOPS survey was approved by the Research Ethics Committee of the University of KwaZulu-Natal. Informed written consent was obtained from all WOPS participants prior to the interviews.The prevalence of arthritis, asthma, cancer, diabetes, heart disease, chronic lung disease, hypertension, and stroke was respectively 31.5, 7.3, 1.7, 10.2, 1.2, 1.7, 52.1, and 31.5%. Regarding cognitive problems, prevalences of mild/moderate and severe/extreme memory complaints were respectively 80.6% and 8.3%, and prevalences of learning complaints were 60.7% and 2.8% respectively. Diagnosed depression was reported by 2.8%, and perceived depression (during last 12 months) was reported by 51.9% of the participants.Compared to men, women had higher rates of memory complaints , learning complaints (mild/moderate 67.4% vs. 40.6% and severe/Eextreme 3.5% vs. 0.9%), diagnosed depression (3.5% vs. 0.9%) and self-reported depression (3.5% vs. 0.9%). No significant differences were observed in the prevalence rates of depression or memory and learning complaints.2), while about two-thirds (64%) were obese. The majority of participants rated their health status as neither good nor bad, with 1.4% reporting very good health and 1.2% very bad health. Men were more likely to rate their health status as very good/good compared to women (35.9% vs 18.4%). The prevalences of single and multimorbidity were 40.5% and 30.8%, respectively.Results of the multivariate regression analyses on the association between multimorbidity with cognitive problems and depression are presented in Using data from the South Africa—SAGE Well-Being of Older People Study (WOPS 2010)—the present study investigated the association between multimorbidity with cognitive problems and depression in a sample of the elderly population in South Africa. Because the data were cross-sectional and not representative of the general population, no causal inference can be drawn regarding the associations. However, the analyses did reveal several important findings. First, multimorbidity appeared to be a relatively common condition, as about thirty percent of the men and women were found to be living with more than one chronic disease. The prevalence of hypertension, more than 50%, was notably high.Apart from hypertension, nearly one-third of the participants had experienced arthritis and stroke. Taken together, these figures reflect a high burden of Non-Communicable Diseases (NCDs) in the sample population. Since we included only eight types of chronic illnesses, the actual prevalence of NCDs might be even higher if a wider range of conditions were to be considered . For exaOf note, the prevalences of most of the chronic diseases were higher among women; women also had more than twice the prevalence of multimorbidity compared with their male counterparts. Similar results regarding gender disparity in chronic disease have been observed in a large number of studies using both younger and elderly subjects ,14,15. TApart from the NCDs, the rates of cognitive disorder and depression also appeared to be higher among women. This finding might reflect the higher rate NCDs among women as revealed in the multivariable analyses of this study. Women with multimorbid conditions had significantly higher odds of having memory complaints, being diagnosed with depression, and of having perceived depression during the preceding 12 months. Moreover, in the regression analysis, it was observed that the association between cognitive disorder and depression with multimorbidity was significant only among women; the association between cognitive disorder and depression was significant only with single morbidity among men. This variation is challenging to explain within the scope of the present study. However, a possible contributing factor might be the tendency of women to report poorer self-rated physical and mental health than men ,17.The interrelation between multimorbidity and physiological health has been assessed by previous researchers in higher-income countries. In Australia, a primary-care cohort study found that the prevalence of depression increased with the number of NCDs, concluding that a dose-response relationship existed between the number of NCDs and depressive symptoms . SimilarThe findings of our study contribute to current knowledge and have potential implications for the creation of new health policy in South Africa. However, there are several limitations. As mentioned earlier, the survey was cross-sectional and therefore cannot find causality between the outcome and exposure variables. The prevalence rates are not generalizable for the elderly population in the country. The variables were self-reported and thereby subject to reporting bias. Moreover, we could not adjust the analysis for multiple medication use (polypharmacy), a circumstance that is common among patients with multimorbidity and is itself associated with adverse physical and mental health outcomes. To date, only a small number of countries have been able to conduct investigations in this area, perhaps owing to the inherent complexity of the task. To our knowledge, there is no publicly available dataset on antipsychotic polypharmacy from any country.In conclusion, the prevalence rates of multimorbidity, cognitive disorder, and perceived depression were high among the participants. Women had higher burdens for most of the chronic conditions, including memory and learning complaints and perceived depression. Finally, multivariate analysis indicated a strongly positive association between multimorbidity with memory complaints and perceived depression among women. Conclusions from the present study need to be verified by longitudinal studies that include larger samples and a wider range of chronic conditions. However, our findings do suggest that the elderly population in South Africa should be the focus of research and policy attention to explain and address the high burden of NCDs and their aggravating effects on mental health status, especially among women.


Repetitive pecking at the feather cover of other birds (FP) is one of the most important welfare problems in domestic birds. It is not only characterized by motor symptoms, but also by an innate vulnerability of the serotonergic system. Moreover, the serotonergic system influences cognitive function. Acute tryptophan depletion (ATD) is a widely used method for studying serotonergic function in mammals and has been recently validated in birds. However, a tryptophan-deficient amino acid mixture has never been tested on groups of birds to impact their social behavior, including repetitive feather pecking, nor has it been given to potentially impact their cognition and motor performance. One hundred and sixty White Leghorn laying hens consisting of two genetic lines divergently selected to perform high (H) or low (L) levels of FP, and an unselected control line (UC), were kept in 10 groups consisting of 4 H, 3 L, and 9 UC genotypes. In a counterbalanced order, half of the groups were first subjected to an ATD treatment, while the other half were first given a balanced control (BC) treatment, and vice versa, after which their feather pecking behavior was observed. The effect of ATD/BC on repetitive pecking, motor performance, and cognition was investigated in a 5-s delayed reward task in an operant chamber with 10 phenotypic feather peckers, 10 recipients of feather pecking, and 10 bystanders (who neither performed nor received feather pecks). ATD given to groups of birds induced gentle, repetitive feather pecking in all genotypes. Following ATD, phenotypic feather peckers performed more poorly during the delayed reward task, as seen by their higher number of repetitive, non-rewarded key, and non-key pecks in the operant chamber. In conclusion, ATD impacted the hens' social behavior by increasing the number of repetitive gentle feather pecks at conspecifics. Furthermore, feather peckers were more likely to peck while waiting for a reward after ATD, suggesting a role for the serotonergic system on cognition in these birds. Birds kept for egg-laying engage in a variety of social interactions. However, some behaviors can become problematic, such as excessive aggressive pecking and feather pecking . FeatherEthologists consider FP to be an expression of frustration arising from a lack of adequate opportunity to perform foraging behavior . HoweverThere is some evidence that humans with the above-mentioned psychiatric disorders also show abnormalities in the serotonin (5-HT) system . AdditioSince 5-HT cannot cross the blood-brain barrier, central 5-HT synthesis depends on the availability and transport of its precursor, TRP, in the brain . TRP comAlthough it was previously shown that the 5-HT system might modulate the social behavior problem of FP in laying hens , to our The second experiment, which involved a modified, delayed reward task in an operant chamber, was aimed at comparing repetitive pecking at a pecking key by individuals categorized based on their phenotypic FP behavior before and after ATD treatment. Human studies have shown that participants perform more poorly with a dysphoric distractor in a proofreading task after ATD treatment, possibly due to ATD causing a shift in attention to the distractor . As suchThis study was approved by the University of Guelph Animal Care Committee before testing .2; 15 cm perch length/bird at 90 cm above the ground; 125 × 31 cm platforms 65 cm above the ground; 120 cm2 nest/bird; 10 cm food trough length/bird; 10 nipple drinkers). Cameras  were mounted on the ceiling above the entrance of each pen. Food  and water were provided ad libitum. The hens were kept in a ventilated, windowless room, on a 14:10 h light:dark cycle, with a light intensity of 25 lx at animal level, and an average daily room temperature of 20°C. The pens were separated by opaque boards to prevent physical and visual contact with birds from other pens   = 1.18, P < 0.5; ATD: 3.07 ± 0.0 vs. BC: 2.98 ± 0.06 pecks/min] nor phenotype  effects, nor interactions  were observed for the number of rewarded key pecks (= rewards obtained).Neither significant dietary treatment   = 24.51, P < 0.0001], where P hens delivered the highest number of 5-s delay key pecks during ATD, and the lowest number of pecks during BC treatment , and vice versa in recipient birds . Bystanders were unaffected by the treatment .The number of 5-s delay key pecks were significantly impacted by the treatment and phenotype interaction [To the best of our knowledge, the present study is the first to examine the effect of ATD treatment on (1) FP behavior of laying hens selected for high and low propensities of FP in a group setting, and (2) the key pecking performance of feather peckers (P), pecking recipients and bystanders in an operant chamber with a delayed reward schedule.In the home pens, ATD treatment increased the overall number of gentle FP bouts for all genotypes of the hens. Severe FP was not triggered at all after ATD compared to the BC treatment. This result supports part of our hypothesis and confirms studies by van Hierden et al.  and SavoNo differences in propensity to develop gentle or severe FP due to ATD were associated with genetic line. We hypothesized that the H genotype, selected for FP activity, would be more vulnerable, due to potential heritable neurochemical deficits , leadingn = 32) within the 4-min testing period was not reached by any of the subjects  and this may give rise to the concern that some of the birds were not completely motivated to obtain rewards. Interestingly, video observations showed that rather than spending 2 s eating from the feeder, some hens learned to spend that time using their beaks to push the food out from the feeder onto the ground, in order to freely consume the food even after access to the feeder was removed. This might explain why the hens did not achieve the maximum number of rewards.Overall, in the operant chamber, rewarded key pecks were unaffected by ATD/BC treatment or phenotype . This indicates that all birds (who were not food-deprived) were similarly motivated to obtain a high-quality food reward, irrespective of treatment. However, the maximum number of theoretically obtainable rewards .AH-M, PB, JK, WK, and PF conceived and designed the study. PB, JC, and PM carried out the study. JC and PM analyzed the videos. PB analyzd the data with help of Dr. Michelle Edwards (UoG statistician). PB and JC drafted the manuscript. AH-M, PM, JK, WK, and PF contributed to writing the manuscript. All authors read and approved the final manuscript.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.


Low birth weight  is an adverse perinatal risk that may reflect a poor intrauterine environment. While LBW has been a well-known predictor of physical, neurological, cognitive and psychological deficits later in life, minimal research has been done on small head circumference and low 5 minute Apgar scores, and their association with subsequent developmental abnormalities.The current study aims to demonstrate that small head circumference and low 5-minute Apgar scores are predictors for developmental abnormalities throughout childhood and later.Using a longitudinal design, 2,151 individuals' physical, neurological, and cognitive functioning in childhood, as well as psychological functioning in adulthood, was assessed as a function of three perinatal risk factors: LBW, small head circumference and low Apgar scores.Similar to findings with LBW, small head circumference or a low Apgar score were associated with increased number of hospital visits (p<0.0001 and p=0.005 respectively) and neurological abnormalities (p<0.0001 and p=0.001 respectively) at age 1. Intelligence quotient (IQ) scores at ages 4 and 7 were significantly lower for those born with small head circumference (p<0.0001) or low Apgar scores (p=0.002). Finally, the incidence of anxiety in adulthood was significantly higher for those born with small head circumference (p=0.03) or low Apgar scores (p=0.004) compared to their counterpart.Small head circumference and low a Apgar score are predictors of later physical, neurological, cognitive and psychological abnormalities, and can complement LBW, a more frequently used perinatal risk factor, and thus be used to screen for future developmental deficits, together with LBW. Low birth weight (LBW), defined as less than 2500 g, has been implicated as a predictor for subtle, but adverse, childhood neuro-cognitive functioning, including cognitive, neurological and psychological challenges -3. IndivWhile not extensive, prior researchers have investigated the effects of small head circumference and low Apgar scores on various functioning of the offspring. For example, small head circumference at birth has been associated with increased vulnerability to the immature brain  lower veThe current study will examine the prevalence of abnormality in neurological development, cognitive functioning and academic achievement, medical problems, and language, hearing and speaking in childhood by the three perinatal risk factors, i.e, LBW, small head circumference, and low Apgar scores, simultaneously. LBW would serve as a reference, i.e, the established surrogate measure, in order to have a base of prediction with which we could compare the head circumference and Apgar scores predictability. Considering multiple indicators of poor intrauterine environment may also help identify infants at greater risk for neurobehavioral development and psychological symptoms in adulthood.Data come from part of the Johns Hopkins Collaborative Perinatal Study. Pregnant women receiving prenatal care at the perinatal clinic delivered their babies at Johns Hopkins Hospital during 1960-1964. Their children were followed until they were 8 years of age. Twenty-five years later, those offspring were re-contacted (mean age 31). Hardy et al.  for a fund generation subjects (G2) eligible for follow-up between 1992 and 1994, 2,344 (79.8%) G2 had information on the three perinatal problems  and an initial neurological examination at 4 months old. Between those who are included in the analysis  and those excluded (n=543), there is no major demographic differences except for the sex of the participants, where a greater proportion of male than female offspring was excluded. The frequency of missing data on neurobehavioral and cognitive function measures ascertained between ages 0 to 8 for dependent variables vary, ranging from 0% to 2.9%. 7.6 to 9.1% of speech, language, and hearing function at age 3, and 10.5% of the health care utility between 4 and 8 years old, and are missing.Of the 2,694 2Of those 2,151 cohorts, 1,540 were located 25 years later. Approximately 91% of the located sample (n=1403) provided their general psychological status. The frequency of missing data for controlling variables was negligible (less than 0.2%).Birth weight, head circumference, and 5 minute Apgar scores were recorded by a nurse observer in the delivery room at the birth of the infant. Mean (sd) of these three perinatal indicators are 3,024 g (561 g), 33.6 cm (4.0 cm), and 8.8 (1.2) respectively. These risk factors were dichotomized at the cut-off points, to create indicator variables for LBW , small head circumference (≤ 32 cm), and low Apgar score (<7), which are conventionally used at clinical setting.The number of medical visits and the number of hospitalizations at ages 1, 4, and 8 year were reported based on caretakers' self-reports.Neurological abnormalities at ages 4 months, 1, and 7 years were evaluated through physical examination conducted by a pediatrician, with special training in neurology, or a pediatric neurologist. Neurological abnormalities included skull size and shape, spinal abnormalities, primary muscle disease, mental retardation, emotional and psychiatric disorders and detected squint. Non-neurological abnormalities included all conditions except neurological abnormities and minor acute upper respiratory infection. Based on observation of 116 items the child's neurological status was characterized as none , suspect, or definite. Finally, the impression of the child's non-neurological status was characterized as none, minor, or questionable/definite.Bayley Scales of Mental and Motor development  was usedSpeech, language, and hearing problems were assessed by a speech pathologist and audiologist, using The Three-Year Speech, Language, and Hearing Examination . The exaDevelopmental profiles were assessed by child psychologists in four areas: fine motor development, gross motor development, concept formation, and behavior. Each area was scored separately and the child's performance was indicated as “normal” or “suspect or abnormal.” Normal was coded as 0 and suspect or abnormal was coded as 1.Fine motor development was measured by Wallin pegboard, coping forms, stringing beads, and porteus maze. Gross motor development was measured by line walk, hopping, and ball catch. Each developmental task was scored pass or fail and established the presence or absence of motor defects first. Concept formation was measured by Graham-Ernhart block sort test, which provided a significant discrimination between brain damaged and non-brain damaged preschoolers. The test consists of sorting materials based on size, shape and color.Cognitive functioning was assessed using the Stanford-Binet IQ  and admiChild psychologists measured intelligence, visual motor coordination, and academic performance. Intelligence was measured using Wechsler Intelligence Scale for Children (WISC) at age 7 . StandarAdult psychiatric status was measured using the General Health Questionnaire-28 (GHQ-28) ,27. DeprSocio-demographic confounders include mother's race and poverty level at delivery and child's sex. Poverty level represents the ratio of the mother's annualized income to the poverty level based on the Social Security Bulletin Annual Statistical Supplement . GestatiFirst, to examine group differences by LBW, small head circumference, and low Apgar scores for rates of offspring problems at different times in infancy, childhood, and adulthood, univariate analyses were conducted using X2 tests for dichotomous outcomes and analysis of variance (ANOVA) for continuous outcomes. The univariate analyses were followed by multivariate analyses to adjust for potential confounders: logistic regression analysis was used for dichotomous outcomes and analysis of co-variance (ANCOVA) was used for continuous outcomes. Potential confounders were considered a priori and were included in the model as covariates for statistical adjustment. All reported p values are two-tailed.Among 2,151 offspring, 82.4% were Black, 17.94% were White, and 49.9% were female. Approximately a quarter of the mothers completed at least high school and approximately 30% had less than or equal to an 8th grade education. With regard to mothers' demographics, the mean (SD) number of progeny, age and individual income during the 1st trimester pregnancy were 3 (2.4), 24.9 (7.1) and $1,022 (515) respectively.Research pediatricians and pediatric neurologists who were blind to perinatal risk status, assessed development of the offspring in three categories  at ages 4 months, 1 year, and 7 years. LBW was associated with a 2-fold increased risk for definite non-neurological abnormality at 4 months and 1 year, but not with minor non-neurological abnormality, and the differences diminished by 7 years of age. There was a significant increased risk for questionable/definite non-neurological abnormality among offspring with small head circumference at 4 months. However, unlike with LBW, the risk for non-neurological abnormality diminished by 1 year of age. There was no notable increased risk of non-neurological abnormality (neither minor nor definite) with low Apgar scores.At the 4 year neurobehavioral development assessment, with an exception of the association between low Apgar scores and concept formation, all of the three perinatal risk factors were associated with a smaller, but significant, increased risk for fine motor , gross motor , intelligence , and concept formation abnormalities The current study evaluated the short- and long-term neuropsychological developmental consequences of LBW, small head circumference and low Apgar scores simultaneously, capitalizing on a longitudinal design. Our findings are generally consistent with previous literature and extend its scope by highlighting four main findings. First, the number of hospital admissions increased for all three perinatal risk factors-LBW, small head circumference, and low Apgar scores-during the first year of life (0-12 months) and the significant difference by LBW persisted until age 8. Second, each of the three perinatal factors was an independent predictor of neurological, non-neurological, cognitive and language difficulties later on in childhood (ages 4 months-8 years). Third, the difference on various indicators of neurobehavioral impairment between offspring with and without a perinatal factor appear to dissipate on its own over the course of the first few years, however, in adulthood there were modest but significant differences in psychological functioning. Lastly, with the exception of a brief period of time in preschool years, small head circumference and low 5 minute Apgar scores are significant independent predictors for suboptimal functioning, even after controlling for the effect of LBW.Our findings on the increased risk of hospital admissions in relation to birth weight status were consistent with previously demonstrated links between LBW and hospital admission ,33. An aThere is a large difference between groups for the number of hospitalizations from ages 0 to 12 months because the severity of the same illnesses is intensified for children with LBW. During early infancy, children born with LBW appeared to be more vulnerable, such as by the presence of compromised immune systems, than their normal BW counterparts thus contributing to a substantial difference between groups (p<0.0001). However, for ages 13 to 48 months, those born with LBW who have survived the first 2 years of life might have caught up with their deficits, including their immune functioning. By age 8 the possibly compromised immune systems of children born with LBW might have been highlighted once again when diseases from school exposure may be more likely.In our study, when the effects of potential confounders, such as sex, race and mother's poverty level during pregnancy, were controlled for, LBW was no longer associated with minor non-neurological abnormalities at 4 months or 1 years of age. Those findings, especially in non-neurological abnormalities among offspring born with LBW, can be explained by malformation in the central nervous system  and loweWe found that neurological development at 8 months and 4 years was significantly affected by all of the three perinatal risk factors we examined. Such distinctions between groups persisted in IQ at ages 4 and 7 as well as in achievement scores at age 8. Readers, however, need to be cautious when interpreting the greater magnitude of effects on development as measured by the Bayley Scales. 22 Recent meta-analysis shows that the Bayley Scales on development of very preterm and very LBW children are limited . AdditioFinally, it is important to be reminded that our findings provide evidence that small head circumference was a significant and even stronger predictor of psychological functioning, such as depression, social dysfunction, and somatic symptoms, and low Apgar scores were associated with an increased risk for anxiety. Although growing evidence show that LBW is associated with adult psychiatric disorders . LBW on Our study has some methodological strength. First, the second generation cohort  was prospectively and systematically followed from birth and studied longitudinally for over 30 years. Our sample came from predominately unprivileged backgrounds of low socioeconomic status with little to no education and a high proportion of ethnic minorities (82.1%). However, they were randomly selected regional  representatives who sought prenatal care at Johns Hopkins Hospital rather than a clinical sample with either serious psychopathology or medical illness. Nevertheless, we acknowledge that generalizability may be limited. Second, birth weight, Apgar scores, and head circumference were recorded by a research nurse at the time of delivery rather than based on mother's retrospective report. We also acknowledge potential limitations. First, we need to interpret our findings in light of the level of obstetric care in the1960's. Our sample was born in the pre-NICU era and the mortality rate for those born very early and with very LBW was higher than it is currently. Second, although we have adult psychological function data, the participants included in this analysis in adulthood is relatively low. While 75% of adult participants were identified and 67% of the original participants were followed, we were unable to include participants who could have survived had they been born in the modern times, who maybe more likely to have various and more serious impairment. Finally, the concept of special or remedial education, and a realization of the need for early intervention, has also advanced considerably since the late 1960s. It may be that with early intervention and special education programs provided at the preschool level, that some of these results may have ameliorated or disappeared. However, there are also many children who did not survive in the 1960s who would today. They may be more involved and thus more likely to demonstrate long term negative sequellae.Despite these limitations, the study contributes to the existing literature by providing evidence that 1) examining different perinatal risks, can improve our ability to predict different signs of neuro-cognitive impairments at different times in a life cycle, 2) that small head circumference which does not resolve by age 2 is a risk factor for additional neuro-cognitive impairments, and 3) that low 5 minutes' Apgar scores is a long term predictor of neurological, cognitive, and psychological development. There is ample evidence that early intervention education services can have positive long-term effects most shown for neurological abnormalities and less so for genetic ones ,42. Gene


No local recurrence was observed during the follow-up period. PRP + HA treatment showed stronger regenerative potential in terms of epidermal proliferation and dermal renewal compared with HA alone.Chronic ulcers are characterized by loss of substance without a normal tendency towards spontaneous healing. The Wound Bed Preparation Guideline advises that after diagnosis, the expert should correct the biological state of the ulcer micro-environment based on TIME principles . There are many ways to treat such ulcers, for example through use of advanced dressings, negative pressure, surgical toilets, dermal substitutes, autologous skin grafting, and free or local flaps. In vitro and in vivo pre-clinical models hold widely acknowledged potential yet complex limitations. Tissue bioengineering could be an ideal approach to foster innovative strategies in wound healing. Our observational study reports on an in vitro and in vivo evaluation of a bio-functionalized scaffold composed of platelet-rich plasma (PRP) and hyaluronic acid (HA) used in 182 patients affected by chronic ulcers (diabetic and vascular), comparing the results with a control group of 182 patients treated with traditional dressings . After 30 days the patients who had undergone the combined treatment (PRP + HA), showed 96.8% ± 1.5% re-epithelialization, as compared to 78.4% ± 4.4% in the control group (HA only). Within 80 days, they had 98.4% ± 1.3% re-epithelialization as compared to 87.8% ± 4.1% in the control group (HA only;  Chronic ulcers are characterized by a loss of substance without a normal tendency towards spontaneous healing. Usually, these lesions have a vascular etiology, with 70–80% being a result of venous pathology, 15–25% due to arterial pathology, and 5–15% resulting from a mixed etiopathogenesis. All other reasons  represent 5% of the total cases. A correct approach is fundamental . The mosPlatelet-rich plasma (PRP) is standard routine for researchers and physicians. Preparation methods and reparative mechanisms are still in the discovery phase due to their various growth factors ,5,6. PRPHyaluronic acid (HA) is an important element in the extracellular matrix (ECM). Its physiological functions relate to its structural role within the ECM and its skill in interacting with cell surface receptors. Its viscous supplementation feature is able to reduce pain and improve tissue viscoelasticity. It is able to hydrate and modulate the cellular microenvironment, while its cell surface receptor bindings induce cell to-cell adhesions, cell-substrate adhesions, proliferations, and cell migrations. Therefore, HA, as a scaffold, not only facilitates the entry of a large number of cells to the wound site but also contributes to the orientation of the ECM  and fibrCombined use with HA and PRP in a bio-functionalized scaffold could supply many advantages for wound healing through faster healing compared with advanced dressing and single use of PRP as a bio-stimulator or HA as a scaffold, with a significant reduction in the costs of hospitalization and reduction of pain in the immediate postoperative period ,10,11,12There are many studies about the combined use of hyaluronic acid and PRP in chronic ulcers ,16,17,18The aim of our study is to evaluate how platelet-rich plasma and hyaluronic acid help healing torpid chronic ulcers. In addition, this paper aims to perform an observational study with standard parameter statistics and comparable control groups using the same tools, methods, centrifugation phase, and quantities of blood, and similar basal levels of platelets in patients.This observational case-series study was conducted following the principles outlined in the Declaration of Helsinki and internationally established ethical principles in clinical research . A qualiIn total, 364 consecutive patients came to our Complex Operational Unit (UOC) at the Plastic and Reconstructive Surgery Unit of the Tor Vergata University of Rome for chronic ulcer treatment from 2010 to 2018. All of them underwent photographic and magnetic resonance imaging (MRI) in a pre and post-procedure examination of the soft tissue in order to evaluate the best way to heal torpid ulcers.In addition, in the more complex cases a high-resolution CT (computerized tomography) scan with three-dimensional imaging for a better view of the anatomical structures was performed. Postoperative follow-up took place at 7 days, 2 and 4 weeks, 3, 6, and 12 months, and then annually. All patients were enrolled following a series of advanced dressings, which allowed for preparation of the wound bed. Intraoperative surgical debridement was performed.Pre-operative data of the patients were acquired including information on age, gender, diagnosis, and comorbidities. The surgical procedure type (only HA or combined PRP + HA), post-operative hospital stay, pathological differentiation, and ulcer stage as well as morbidity were analyzed in order to compare the surgery risks and benefits in the two groups.Patients were divided in two groups: Group 1  and Group 2 .Eligibility criteria  were ideThe authors also considered exclusion criteria. Exclusion criteria were divided in two types: local and systemic. The systemic criteria included cancer, autoimmune diseases, chemotherapy or radiotherapy, treatment with antiplatelet agents, platelet disorders, thrombocytopenia, anti-aggregating therapy, bone marrow aplasia, uncompensated diabetes, and sepsis. The local criteria included osteomyelitis, and loss of substance representing more than 50% of the segments. The authors did not considerate smokers, compensated diabetes, or genetic disorders as exclusion criteria.®) preparation involved centrifuging a patient’s blood sample (10 cc). A vial of blood was placed in a centrifuge, where it was rotated at intensely high speeds for 10 minutes at 3000 rpm. PRP as a bio-stimulator was utilized topically with intra-lesional and perilesional infiltrations in the treated area (® Centrifuge) for another 15 minutes. This elastic, gelatinous, and soft cloth PRP gel was applied to the wound bed (6/µL + 20%)Evaluation of the number of platelets ;Volume of PRP obtained (variable according to the type of use and the size of the ulcer);Labeling of each sample of PRP for each patient for laboratory control;Adverse reaction signaling (did not occur).The surgical technique followed the protocols of the Plastic and Reconstructive Surgery Unit at the Tor Vergata University of Rome ,10. The ted area B activatound bed D. This P®) was the biological dressing for tissue regeneration.At the end of procedure we applied the HA scaffold C,D in orHA can remain in the site for 15–20 days when growth factor action peaks; therefore, use of drug therapy can be reduced, which may be of advantage to the patient. Elastic compression was applied in order to avoid venous-lymphatic interference with the regeneration process. During the treatment, antibiotic therapy was administered in accordance with the results of culture through antibiograms, and continued for 7 days. For all patients, we followed the following steps: (1) After 7 days the secondary dressing (gauze) was changed; (2) For both groups, the silicone film  was removed; (3) After 5 weeks, for Group 1 (HA only) the excess of HA was removed and replaced as necessary; (4) After 30 days, healing or apposition of new HA was applied.The following rating system was used in order to evaluate wounds condition at 15, 30, 60, and 90 days after the treatment. We assigned a number grade based on lesion depth, with 0 = closed wound, 1 = superficial cutaneous lesion interesting epidermis, 2 = deep skin lesion interesting epidermis and derma, 3 = exposed bone and /or tendon (depending of the site of the sore), and 4 = exposure bone and/or tendon with infection.Incisional punch biopsies of ulcers (3 mm in diameter) were obtained at baseline (pre-treatment) and post-treatment (4 weeks). Microscopic evaluation of routinely haematoxylin & eosin-stained paraffin sections ,21 was pt-test to investigate the healing rate of wound healing in terms of re-epithelialization over the time. Differences were considered significant if p-values were < 0.05.The categorical data are presented as frequencies and percentages, recurrence rates after the PRP procedure with those after HA using Kaplan–Meier survival analysis, and Student’s In terms of wound features, the classification was based on their dimensions, the depth, the size the area, and anatomical site distribution . In totaFinally, regarding the anatomical site distribution , 45.05% Patients in the two study groups were comparable for age, indications, and ulcer grade. In total, 182 patients were enrolled and evaluated in this observational study. Overall, 52% were males and 48% were females.p < 0.05). Postoperative pain assessment was done in patient samples (To assess pain severity in patients we used a survey which was developed based on the available literature and our experience. No difference was noted for significant pain (pain score ≥ 3) during the procedure and one day after treatment. Discomfort pain (pain score ˂ 3) was more frequent in Group 1 (HA only) than in Group 2 (PRP + HA) 7 days after procedure ( samples .p < 0.05) over the time. In about 30 days, 73.62% of patients reached complete healing depending on the wound size, wound depth, and general patient condition. Note that most patients from both groups reached complete healing over 30 days. Patients who underwent combined treatment healed in a shorter time  patients healed better than other groups . In addiOverall, in Group 1 (HA only) the healing process in the first 90 days was quicker, but after that the therapy with HA alone was not sufficient. Patients from this group, in fact, needed a second surgical procedure. The arrow in p < 0.01). The in vivo result of a patient affected by a necrotic ulcer . Only 1.6% of Group 2 (PRP + HA) patients who presented an over 20-cm2 wound size and an over 1-cm wound depth reached complete healing over 45 days. The median duration to complete healing was 45 days. No local recurrence was observed during the follow-up period.Within 80 days, Group 2 patients had 98.4% ± 1.3% re-epithelization compared to 87.8% ± 4.1% in Group 1 .Representative microphotographs of haematoxylin & eosin staining are reported in Based on this study it can be stated that PRP combined with HA, in general, stimulates a synergistic enhancement process of tissue repair because of its similar logical and mechanism action. Furthermore, it allows a total recovery and bioavailability of the platelet growth factors, is biologically safe (not immunogenic), controls inflammation, reduces pain, and has hygroscopic, rheological, and viscoelastic properties. The combined technique acts as scaffold for cellular growth and reduces healing times, costs, and hospitalization times.Successful completion of this trial will provide evidence of the best treatment strategy for patients with chronic ulcers. Results from this study can lead to a better understanding of the role of HA as a scaffold and that of the combined technique with PRP as a bio-stimulator in chronic ulcer treatment ,14. OverThe proliferative phase of wound healing, known as the granulation phase, is marked by angiogenesis, fibroplasia, and extracellular matrix (ECM) deposition, all leading to re-epithelialization. The remodeling phase, also known as the maturation phase, is the final stage of wound healing after granulation and wound re-epithelialization or skin desquamation. During this wound repair stage, ECM is deposited and remodeled .Chronic ulcers do not heal spontaneously few months . GeneralWound healing is an evolutionarily conserved complex multi-cellular process that in skin targets barrier restoration. During normal tissue repair in vivo, platelets release high concentrations of biologically active proteins including growth factors . They arPRP is an autologous platelet concentrate contained in a small volume of plasma ,35, enriThe acquired process must be sterile and accurate. It is relatively economical, easy, and minimally invasive. Therefore, use of platelet-rich plasma is a simple and safe procedure . AnotherHA is a biopolymer present in the extracellular matrix of skin, cartilage, bone, and brain, among other tissues . HA is aDuring the tissue regeneration process a number of cells appear, favoring new tissue formation and reconstructing ECM assembly in order to support cellular proliferation and differentiation for the restoration induced previously. Therefore, HA facilitates entrance of a large cells number into the damaged area and supports the orientation of the ECM fibrous component .Intra-articular HA injections are widely accepted for the treatment of pain associated with osteoarthritis (OA) . CurrentHA acts as a scaffold for the PRP . It is aThere are many studies suggesting regenerative medicine procedures associated with next-generation biomaterials could be used to restore an appropriate environment that encourages healing ,21,44. OThe authors’ goals are to elaborate a cellular mechanism approach in order to regenerate and promote one’s own natural growth factor release. This study could demonstrate that combined treatment with PRP and HA as a bio-functionalized scaffold can be used to cover soft tissues deficits without requiring frequent dressing changes. Their combination decreases edema and chronic pain ,49,50,51A previous study reported the use of PRP and HA for pressure ulcers , post-suThis study reveals that combined treatment with PRP and HA optimizes granulation formation and tissue regeneration. The combination thus provides more rapid wound closure with an excellent aesthetic improvement. This synergetic performance allows for tissue regeneration because of their analogous action mechanisms. Both are involved in the pathophysiological mechanisms underlying wound healing .Moreover, this combination helps to prevent infection, has an algiatry effect, and it is able to interact with ECM and cells forming a cell growth scaffold. This technique is minimally invasive and well tolerated by patients. In Group 2 (PRP + HA) patient granulation tissue formed faster when compared to Group 1 (HA only). Healing time was shortened: within 30 days 73.62% (± 2.1%) reached complete re-epithelization  and the requirement for further surgical treatment was reduced depending on the wound size and depth and general patient condition. Regenerated tissue demonstrated a notable improvement in terms of texture and a shorter healing time, with good cutaneous elasticity, a significant pain decrease ,51 immedIn order to prevent possible complications, absorbable surgical suture use during interventions as well as collagen-based elastin and biomaterials available mainly for very deep and large wound sizes should be considered.Our results provide evidence that PRP and HA are useful in therapeutic measures for open wounds. However, the relationship between in vitro and in vivo benefits and platelet concentration remains unclear due to inter-individual variability and the particular factors influencing each case .This study reveals that HA has a durable effect after 6 months. Group 2 patients (treated with PRP + HA) presented an amplified effect of both components. In addition, PRP has an anti-inflammatory power and it acts much more rapidly than hyaluronic acid alone. Thus, combined treatment is painless if compared with treatment with HA alone. Treatments can be repeated only when there are no contraindications, including infection in rare cases.p < 0.05) and patient pain, and is cost-effective compared to traditional treatment, increasing the patient’s quality of life.This study concludes that HA acts as a scaffold for the PRP. The speedy development of granulation tissue can help to shorten the healing times and reduce the need for reconstructive surgery on supplementary soft tissue. Therefore, body repair mechanisms are stimulated to heal previously irreparable tissues. In addition, the study validates that combined treatment with PRP as a bio-stimulator and HA engaged in a bio-functionalized scaffold can reduce the healing period (For that reason, combined use with PRP and HA is a positive method for chronic ulcer treatment both from economic and surgical perspectives.Additional studies to determine the ideal PRP concentration for optimal in vitro and in vivo benefits in order to standardize scientific literature would be recommended.


Background and objectives: High-flow nasal cannula (HFNC) can be used as a respiratory support strategy for patients with acute respiratory failure (ARF). However, no clear evidence exists to support or oppose HFNC use in immunocompromised patients. Thus, this meta-analysis aims to assess the effects of HFNC, compared to conventional oxygen therapy (COT) and noninvasive ventilation (NIV), on the outcomes in immunocompromised patients with ARF. The Pubmed, Embase and Cochrane databases were searched up to November 2018. Materials and Methods: Only clinical studies comparing the effect of HFNC with COT or NIV for immunocompromised patients with ARF were included. The outcome included the rate of intubation, mortality and length of stay (LOS). Results: A total of eight studies involving 1433 immunocompromised patients with ARF were enrolled. The pooled analysis showed that HFNC was significantly associated with a reduced intubation rate (risk ratio (RR), 0.83; 95% confidence interval (CI), 0.74–0.94, I2 = 0%). Among subgroup analysis, HFNC was associated with a lower intubation rate than COT  and NIV , respectively. However, there was no significant difference between HFNC and control groups in terms of 28-day mortality , and intensive care unit (ICU) mortality . The ICU and hospital LOS were similar between HFNC and control groups . Conclusions: Use of HFNC may decrease the intubation rate in immunocompromised patients with ARF compared with the control group, including COT and NIV. However, HFNC could not provide additional survival benefit or shorten the LOS. Further large, randomized controlled trials are needed to confirm these findings. With the aggressive surveillance and the improvement of medical care, the incidence of cancer, as well as the survival of cancer patients, are both increasing steadily ,2,3. TheAll clinical studies were identified by a systematic review of the literature in the PubMed, Embase, and Cochrane databases until 10 November 2018, using the following Mesh terms—“high-flow nasal cannula”, “nasal high flow”, “humidified high-flow nasal cannula”, “respiratory failure”, “acute hypoxemic respiratory failure” and “acute respiratory failure”. Only randomized controlled trial (RCT) or observational studies that compared the clinical efficacy of High-flow nasal cannula (HFNC) and conventional oxygen therapy (COT) or noninvasive ventilation (NIV), for immunocompromised adult patients with acute respiratory failure (ARF), were included. Immunocompromised status was defined as a chronic use of steroids, the use of other immunosuppressant or chemotherapeutic agents, solid organ transplantation, solid and hematologic malignancy, human immunodeficiency virus (HIV) infection, or primary immune deficiency. In addition, we searched all references in the relevant articles and reviews for additional eligible studies. We excluded case reports or case series, single arm studies, studies enrolling pediatric patients on neonate, and conference abstracts. Two reviewers (Chang and Wang) searched and examined publications independently to avoid bias. When they had any disagreement, another author (Lai) resolved the issue in time. The following data included authors, year of publication, study design and duration, sites of study, the demographic characteristics of the study population, immunocompromised conditions, disease severity, indication of oxygen therapy for ARF, and the outcomes. Neither ethics board approval nor patient consent was required, due to the nature of a systematic review. The Cochrane Risk for Bias Assessment tool  and modiQ statistic generated from the χ2 test. The proportion of statistical heterogeneity was assessed by the I2 measure. Heterogeneity was considered as significant when its p-value was less than 0.10, or the I2 more than 50%. The fixed-effect model and the random-effects model were applied when the data was homogenous, and heterogeneous, respectively. For dichotomous outcomes, we estimated the summary risk ratio and 95% confidence interval (CI); for continuous data, we estimated summary mean difference (MD) and 95% CIs. The mean and standard deviations were estimated from the median and interquartile ranges according to a previous study [p-value <0.05 was set as the threshold of statistical significance. Sensitivity analyses were conducted by excluding or subgrouping studies to reduce the potential confounding effects.The primary outcome was the rate of intubation. Secondary outcomes included all-cause mortality, including 28-day, intensive care unit (ICU) or hospital mortality and length of stay (LOS) in the ICU and hospital. The statistical analysis was conducted using the software Review Manager, Version 5.3. The degree of heterogeneity was evaluated with the us study . A p-valThe search program yielded 246 references, including 53 from Pubmed, 169 from Embase and 24 from the Cochrane database. Then, 195 articles were screened after excluding 51 duplicated articles. Finally, a total of eight studies ,29,30,31I2 = 0%, I2 = 0%) and NIV , respectively (I2 = 21%). In contrast, no difference was found in two pooled RCTs  and two post-hoc analyses of RCTs  (I2 = 21%), but no difference was found in four multicenter studies .Among all of the eight enrolled trials, the intubation rate was 37.6% (273/727) and 45.3% (324/716) in the group of patients who were assigned to HFNC and controls, respectively. The pooled analysis showed that HFNC was significantly associated with a reduced intubation rate  . In the I2 = 48%, I2 = 57%, I2 = 69%; hospital LOS: Mean difference, −0.12 days; 95% CI, −1.86 to 1.61, I2 = 64%)  .p <  0.00001; I2 = 30%) and extubation failure  when compared with COT in a meta-analysis [I2 = 49) when ARF patients were treated with HFNC therapy, compared with COT [This meta-analysis enrolled a total of eight studies involving immunocompromised patients with ARF and provided several significant findings. Most importantly, the use of HFNC can help reduce the intubation rate in immunocompromised patients with ARF. The effect remained significant in the subgroup analysis while compared with COT and NIV. The similar findings have been reported in previous studies  in otheranalysis  of 18 RCwith COT . Even fowith COT  showed tHowever, the significant effect of HFNC in reducing the intubation rate was only evident in the pooled analysis of four retrospective studies or single center investigations. In contrast, no significant difference was noted in the pooled analysis of two RCTs, two post-hoc analysis of RCTs, or four multicenter studies. This may be explained by the different study design. In the single center, the treatment protocol may be more consistent than in the multicenter RCT trial. In addition, none of the studies were double-blinded clinical trials, due to the nature of this kind of study. The lack of blinding may affect the treatment and the following assessment of the outcomes. Although our finding seems robust due to the low heterogeneity in the analysis of primary outcome, we still needed more solid data from large scale RCTs to confirm our findings.Despite finding a positive impact of HFNC on reducing intubation rates among immunocompromised patients with ARF, there was no significant difference between HFNC and control groups regarding secondary outcomes, including 28-day mortality or ICU mortality, and length of ICU or hospital stays. Although the level of evidence regarding our findings of secondary outcomes was low due to the relative high heterogeneity and limited RCTs, these findings can be explained by that the causes of ARF, and the characteristics of study populations are protean, and the treatment of ARF is complicated. As a lone intervention, HFNC cannot improve overall outcomes including mortality and LOS. In addition, HFNC might not reduce mortality (despite reducing intubation rates) because of potential harm caused in the patients that were eventually intubated. In these patients, intubation may have been delayed, and outcomes made comparatively worse.I2 value of more than 50% in the secondary outcome analysis. These heterogeneities could be caused by some significant variations in study design, and the clinical characteristics of enrolled patients. Second, most of the participating ICUs were located in Europe, especially in France, so the generalizability may be limited.Although this meta-analysis is updated, and includes double the number of patients than the previous meta-analysis, there were several limitations within this study. First, there was relatively high heterogeneity with an This meta-analysis suggested that the use of HFNC may decrease the intubation rate in immunocompromised patients with ARF compared with the control group, including COT and NIV. However, HFNC could not provide additional survival benefit or shorten the LOS in the ICU or hospital. Further large, high-quality, randomized, multi-center trials are needed to confirm the effects of HFNC among immunocompromised patients with ARF.