Milk and yogurt are important elements of the human diet, due to their high nutritional value and their appealing sensory properties. During milk processing  and further yogurt manufacture (fermentation) physicochemical changes occur that affect the flavor and texture of these products while the development of standardized processes contributes to the development of desirable textural and flavor characteristics. The processes that take place during milk processing and yogurt manufacture with conventional industrial methods, as well as with innovative methods currently proposed , and their effect on the texture and flavor of the final conventional or probiotic/prebiotic products will be presented in this review. Milk and dairy products have been consumed since the domestication of mammals; yogurt and similar fermented milk products in particular are thought to originate from the Middle East. The original production of fermented milk products derived from the need to prolong the shelf life of milk instead of being disposed . Yogurt Yogurt is defined as the product being manufactured from milk—with or without the addition of some natural derivative of milk, such as skim milk powder, whey concentrates, caseinates or cream—with a gel structure that results from the coagulation of the milk proteins, due to the lactic acid secreted by defined species of bacteria cultures. Furthermore, these bacteria must be “viable and abundant” at the time of consumption . The aboYogurt manufacture begins with the milking of the mammal, includes several processes, ending with the packaging of the final product, yogurt. The aim of this work is to present the processes that take place in the dairy industry; no further details about the milking and transportation of the milk will be included, even though these critical stages affect the quality and safety of the final product. Yogurt is mainly produced from bovine milk, although milk from other mammals is utilized for yogurt production as well. Yogurt derived from the milk of species other than bovine tends to vary in several sensory and physicochemical characteristics, due to differences per milk composition. For instance, yogurt derived from milk with high fat content  has a more creamy texture compared to that derived from milk with lower fat content . Therefore, the species of the milk-producing mammal significantly influence the characteristics of the produced yogurt .Raw milk undergoes, in the dairy industry, centrifugal clarification to remove somatic cells and any other solid impurities . AfterwaThe standardization of milk refers to the standardization of fat and solid-non-fat content (SNF). Bovine milk fat content varies from 3.2%–4.2% w/w. The fat content of the milk is adjusted to range from <0.5%, for skim milk, to 1.5%–2%, for semi-fat milk, to 3.5% for full fat milk. As far as yogurt is concerned, the fat content ranges from 0.1%–10% according to consumer demands. In practice, to achieve the designed fat level, either the addition of skim milk or milk fat or the separation of fat from milk via centrifuge and mixing milk fat with skimmed milk is carried out . Also, tThe term of standardization is also applied to the SNF content of the milk. The SNF components of milk mainly consist of lactose, protein and minerals; SNF content of milk varies from 11% to 14% of the total weight of the milk while the SNF of yogurt ranges from 9% to 16%. The SNF content of milk used for yogurt manufacture is altered, in some cases, by producers in order to attain the desired characteristics of the coagulum; the higher the SNF level, the higher the resulting yogurt’s viscosity and firmness. The addition of native milk components is permitted to yogurt and fermented milk products in some countries. It is quite common in yogurt manufacturing to fortify the milk mixture with milk powder (skimmed or full fat), whey protein concentrates or casein powder, to achieve the desired SNF content and subsequently an increase in firmness and cohesiveness . It mustet al. [Milk is a typical oil in water (o/w) emulsion with milk fat globules (MFG) acting as the oil droplets and the milk fat globules membrane as the emulsifier. However, because of the reaction of agglutinins and interfacial tension, the fat globules tend to collide, either by sharing the membrane or because of the Laplace principle, according to which the pressure is greater inside small globules than inside large globules and, hence, there is a tendency for large fat globules to grow at the expense of the smallest. This phenomenon, in addition to Brownian motion, forces the milk fat to rise to the surface of the milk and thus creates the undesirable effect of separation ,10. In oet al. , the smaet al. , while tet al. ,15.2S, methanethiol, dimethylsulphide and carboxylsulphide. These sulfur containing molecules are responsible for the “cooked” off flavor developed during UHT and high temperature pasteurization [Heat treatment of milk is carried out to ensure the safety of the product, whether it is milk itself or any other dairy product, and to exploit several effects that increased temperature has on certain milk components facilitating further processes for dairy products manufacture . Heat trrization . It shourization ,4.Mycobacterium tuberculosis, Coxiella burnetii, Staphylococcusaureus, Salmonella species, Listeria monocytogenes, and Campylobacter jejuni. These microorganisms are killed by even mild heat treatment ensuring that processed milk is safe for consumption. The claim that milk is safe after a mild heat treatment might sound frivolous, but most high-heat resistant pathogens either do not occur in milk  or are outnumbered by other native microorganisms , or cause spoilage before their quantity is enough to cause health issues .Pathogens that can grow in milk, due to bad hygiene practices or hardware failure during the stages of processing, include 2 and O2, an increase in the amount of insoluble colloidal calcium phosphate, a decrease in calcium cations, and forces lactose isomerization, degradation and Maillard reaction, thus affecting the pH of the milk and flavor. Finally for yogurt, the most important changes during heat treatment of milk concern the milk proteins; the reactions of milk proteins, during heat treatment, have a serious impact on the yogurt curd formation and will be described more thoroughly [In addition to the reduction or complete extermination of microbiological load, heat treatment causes release of COoroughly .s1-, αs2- and β-caseins stabilized by κ-casein molecules held together by calcium and calcium phosphate. This structure is stable and requires a high amount of energy to be disrupted. On the other hand, whey proteins in solution have a globular shape. Whey proteins, due to their structure, are fairly stable and do not interact with fatty molecules, calcium ions or caseins in their native state. However, in the case of whey proteins  over 80 °C, their peptide chains unfold, thus denaturating irreversibly. This deformation of the peptide chains exposes their thiol groups and enables them to interact with other molecules forming S–S bonds. Depending on the pH of the environment and the proximity of molecules available, whey proteins can form bonds with other whey proteins and caseins (κ- and αs1- mostly) and also they can be incorporated at the MFG membrane. The denatured whey proteins, especially at pH values lower than 6.5, have the tendency to associate with casein micelles [The casein molecules in milk are in the form of micelles or aggregates of submicelles which are formed from αmicelles ,11,16. Amicelles . Table 1Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in abundance. However, yogurt starter cultures may include other microorganisms as well, like Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus jugurti, Lactobacillus helveticus, Bifidobacterium longum, Bifidobacterium bifidus and Bifidobacterium infantis. Streptococcus thermophilus subsp. thermophilus (ST) is the only species in the streptococcus genus that is used in dairy starter cultures. ST is Gram positive and usually considered thermophilic, however, as the optimum temperature for its growth is 35–53 °C; therefore, ST can be considered as “thermotolerant”. Its cells are spherical in shape, forming chains, during the early stage of their lives and as they mature develop a more rod-like morphology and favor colonial growth. Lactobacillus delbrueckii subsp. bulgaricus (LB) is rod-shaped, Gram-positive, anaerobic bacteria and its optimum growth temperature is 40–44 °C. LB can produce very high amounts of lactic acid by metabolizing lactose [The fermentation process is the most important stage of yogurt manufacture. During this stage, the yogurt curd is formed, and its textural characteristics and distinct flavor are developed ,5. The k lactose . These t lactose .8 to 1010 CFU g−1. The carbonyl compounds formed during fermentation are mostly lactic acid, acetaldehyde, dimethyl sulfide, 2,3-butanedione, 2,3-pentanedione, 2-methylthiophene, 3-methyl-2-butenal, 1-octen-3-one, dimethyl trisulfide, 1-nonen-3-one, acetic acid, methional, -nonenal, 2-methyl tetrahydrothiophen-3-one, 2-phenyacetaldehyde, 3-methylbutyric acid, caproic acid, guaiacol and benzothiozole. These compounds contribute to the distinctive flavor of yogurt. As far as the lipids are concerned, several free fatty acid molecules are liberated due to lipase activity, mostly stearic and oleic acid. The only change in vitamin content is an increase in Vitamin B, throughout the fermentation process and storage. Finally, the quantity of minerals in yogurt remain the same as in milk, the only change is that, due to pH lowering, these minerals are in ionic rather than colloidal form [The growth of the symbiotic culture induces changes in the native components of the milk that are responsible for the physicochemical and sensory characteristics of yogurt. During fermentation, lactose, milk proteins and microbial content, as well as several carbon compounds, suffer major changes, whereas minor changes occur for vitamins and minerals. Lactose is reduced by 30% and produces double the molar amount of lactic acid. Proteins (caseins and whey) aggregate, increasing the consistency of yogurt. Due to proteolysis caused by starter culture, amino acids (mainly proline and glycine) are released into the yogurt, even during storage at 4 °C . During dal form ,18.The concentration of lactic acid in milk during fermentation increases, pH decreases, therefore the carboxyl groups dissociate, serine phosphate is ionized, and the negative charge between casein micelles is increased. However, the presence of calcium phosphate neutralizes this negative charge, keeping electrostatic repulsion down to a level where attractive forces between the protein molecules are dominant. Due to these attractive forces, the casein micelles aggregate and eventually coagulate into a network of small chains; this is responsible for the increase of viscosity and formation of the yogurt coagulum ,19,21,220, pH∞ = initial and end values of pH, respectively; μpH (min−1) = maximum rate of pH decrease; λpH (min) = duration of pH lag phase. Furthermore, the model that describes the evolution of viscosity during fermentation is the modified Gomperz model of Soukoulis, et al. (2007) (Equation (2)) [α0, μα∞ (Pa·s) = initial and end values of viscosity respectively; μv (min−1) = maximum rate of viscosity decrease; λv (min) = duration of viscosity lag phase.The milk fermentation process of yogurt can be described adequately by the evolution of pH and viscosity with respect to time; the model that expresses the evolution of pH during fermentation time is the modified Gompertz models of de Brabandere and de Baerdemaeker (1999) (Equation (1)) :(1)pH0t al. 200  involves the application of pressures from 100 to 1000 MPa. UHP utilization in food products was initiated during the early 1980s and is a non thermal pasteurization method. Studies have shown that milk treatment with pressures of 400–600 MPa for 10 min at 25 °C can achieve a similar result to low temperature pasteurization in terms of pathogenic and spoilage microorganisms inactivation [et al.  and Law, [et al.  describe [et al. . However [et al.  observedet al. [The application of UHP in milk acid gel formation improves the texture and firmness, reduces syneresis and increases the water holding capacity in comparison to conventional yogurts . The comet al.  to increet al. [et al. [et al. [et al. [et al. [et al. [et al. [n-hexanal, n-heptanal, 2-butanone, acetone, dimethylsulfide and chloroform are emitted. The aldehydes can be produced from the breakdown of hydroperoxides generated by photo-oxidation induced by US, whereas the series of C6–C9 1-alkenes could arise from pyrolytic cleavage of fatty acid chains. The benzene formation may be attributed to cleavage of side chains of amino acids such as phenylalanine. These volatiles cause a rubbery and burned aroma [Ultrasound (US) is a sound wave with a frequency higher than the upper limit of human hearing, typically higher than 20 kHz. US has been utilized in the food industry since the late 1960s, for cleaning, monitoring and food component characterization. High intensity US (power level higher than 10 W), when propagated through a solution, generate immense pressure, temperature and shear gradients and thus cause cavitation ,32. Theret al.  and Nguyet al.  showed t [et al.  and Chan [et al.  to cause [et al.  and by C [et al. . US trea [et al. ,37. Rien [et al.  combined [et al. . Further [et al. . Finally [et al. , when mied aroma .et al. [et al. [−2) and showed that the apparent viscosity, yield stress, and viscoelastic properties of yogurt were increased in addition to its structure being strengthened. The increased viscosity and texture of yogurt derived from ultrasonicated milk can be attributed to the denaturation of whey proteins and association of the latter with caseins. Denatured whey proteins are more susceptible to association with casein and casein micelles. Additionally, during acidification the denatured whey proteins, associated or not with casein micelles, aggregate due to the reduction of the repulsive charge. Therefore, denatured whey proteins associated with casein micelles could act as bridging material between casein micelles and as a result the bonds that form in the yogurt matrix are formed more easily, resulting in stronger yogurt coagulum [The implementation of US treatment on the production of fermented dairy products has been studied with promising results. Milk gels and yogurt produced from milk treated by high intensity US have shown improved physical properties and high value of texture characteristics . Increased amplitude level US treatment  significantly improved the water holding capacity of yogurt and increased viscosity and reduced syneresis; moreover, higher US intensity and a higher US exposure time of the milk resulted in increased yogurt viscosity . Increaset al.  even froet al. . A study [et al.  combinedcoagulum .® is an apparatus that causes homogenization via shear, turbulence and cavitation. Initially, it accelerates the fluid and separates it into two microstreams that intersects in a chamber and collide. The impact causes intense turbulence and cavitation and thus the homogenization effect is achieved [et al. [The Microfluidizerachieved ,44. In t [et al. , the mic [et al. . The app [et al. . However [et al. ,46.et al. [Pulsed electric field (PEF) treatment emits intense electric pulses through a continuous medium, to inactivate microorganisms with the best results achieved in fluids. PEF has been applied in dairy systems combined with probiotic cultures . The intet al.  combinedet al. . Despiteet al. ,52.7–108 CFU/g or mL, if not participating in the fermentative process, and if participating, they can reach a concentration between 108 and 109 CFU/g or mL after the fermentative process [Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus helveticus, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis ssp. lactis and Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium bifidus [Modern nutritional probiotic products are classified as those that contain probiotic microorganisms. Probiotic microorganisms are defined as live microbes, which when ingested benefit the health of the host through their effect on the intestinal microflora. In addition, probiotic cultures must be able to survive throughout the intestinal tract, resist acidic conditions during gastric passage and bile digestion. To have their beneficial effect on the health of the host, probiotic strains must, at least temporarily, establish themselves among the natural microflora of the intestine. The initial mention about microorganisms that benefit the health of host being, while delivered via food consumption is attributed to Élie Metchnikoff in the early years of the 20th century . The ter process . The mos bifidus .Fermented dairy products, and especially yogurt, are ideal carriers for probiotic cultures to enter the human digestive system and ensure their survivability through the stomach ; therefoet al. [et al. [Bifidobacterium lactis and Lactobacillus acidophilus resulted in no difference in the sensory evaluation of low and full fat probiotic and non-probiotic yogurt. The same results were noted by other researchers as well [et al. [Bifidobacterium animalis ssp. Lactis) and fortification of WPC (whey protein concentrate) resulted in a stronger coagulum and increased firmness and adhesiveness values for the yogurt. This was attributed mostly to the effect of the fortification with WPC and not to the presence of the probiotic culture.Most probiotic bacteria have no significant effect on the fermentation process or on the yogurt sensory properties . Based oet al.  and Atun [et al. , the add as well ,64. Howe [et al.  the combOverall, the incorporation of probiotic bacteria in yogurt manufacture is viable and this claim is shown by the amount of probiotic yogurt products available in the market; however, the actual challenge is to ensure that the probiotic culture reaches the intestines of the consumers alive and is able to establish itself among the native microflora .trans-galactooligosaccharide, lactulose, isomalt and oligofructose. They have been shown to stimulate the growth of endogenous Bifidobacteria, and make them the predominant species in human feces [et al. [Streptococcus thermophilus or Lactobacillus bulgaricus. However, the end product of this endeavor was characterized as a weak gel with thixotropic and pseudoplastic behavior. Finally, the oligofructose-fortified yogurts had a fairly high acceptance by consumers. Another study conducted by Pimentel [According to Gibson and Roberfroid , prebiotan feces . Based o [et al.  who addePimentel  suggesteStreptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Modern dairy science and nutrition have suggested the involvement of probiotic cultures and prebiotic ingredients in order to increase the nutritional value of dairy products, while minimizing detrimental effects on the sensory characteristics.Dairy processing and yogurt manufacture utilize several scientifically interesting procedures such as centrifugation, homogenization, heat treatment, and in the case of yogurt, manufacture and fermentation. Each procedure significantly affects the quality and sensory characteristics of the final product, whether it is milk or yogurt. Conventional heat treatment includes thermalization, low and high temperature pasteurization, ultra heat treatment and sterilization. The application of heat treatment in milk affects the flavor, the microbial content and the milk proteins. The more intense the heat treatment is, the more radical the changes that occur. Heat treatment also affects the texture of the produced yogurt, increasing the value of its texture characteristics  and viscosity. Homogenization, typically used in dairy processing, is through application of pressure, reducing the milk fat globule size and preventing fat separation from the milk. Other treatments that cause the same homogenization effect with pressure in milk are ultra high pressure, ultrasound, microfluidization and pulsed electric fields. Each type of homogenization causes additional effects on milk and on the produced yogurt. Finally, conventional fermentation process, in yogurt manufacturing, included the utilization of the species


Osmanthus fragrans is an ornamental plant of substantial commercial value, and no genetic linkage maps of this species have previously been reported. Specific-locus amplified fragment sequencing (SLAF-seq) is a recently developed technology that allows massive single nucleotide polymorphisms (SNPs) to be identified and high-resolution genotyping. In our current research, we generated the first genetic map of O. fragrans using SLAF-seq, which is composed with 206.92 M paired-end reads and 173,537 SLAF markers. Among total 90,715 polymorphic SLAF markers, 15,317 polymorphic SLAFs could be used for genetic map construction. The integrated map contained 14,189 high quality SLAFs that were grouped in 23 genetic linkage groups, with a total length of 2962.46 cM and an average distance of 0.21 cM between two adjacent markers. In addition, 23,664 SNPs were identified from the mapped markers. As far as we know, this is the first of the genetic map of O. fragrans. Our results are further demonstrate that SLAF-seq is a very effective method for developing markers and constructing high-density linkage maps. The SNP markers and the genetic map reported in this study should be valuable resource in future research. Osmanthus fragrans Lour. 2n = 46) is a woody, evergreen shrub or small tree species in the Oleaceae family. It is widely distributed and owns cultural significance in China, and was introduced into Europe in the late eighteenth century  status for O. fragrans cultivars in 2004. O. fragrans cultivars are divided into four groups depending on their flowering season and corolla coloration. Cultivars of the Asiaticus Group, Albus Group and Luteus Group bloom only in autumn, while cultivars of the Aurantiacus Group can flower for several times in different seasons except summer , a mainstream molecular marker, was identified as the most suitable for construction of a high-resolution genetic linkage map due to the abundance and ubiquity of SNPs in living organisms , an approach basing on NGS, was recently reported  sequencing technique enable greater specificity and accuracy of SLAF-seq compared to other NGS-based methods, such as complexity reduction of polymorphic sequences (CRoPS)  fine mapping of some important traits in O. fragrans.The objective of this study is to construct the first genetic map for Two cultivars, “Wan Yingui” (Albus Group) and “Huangchuan Jingui” (Luteus Group), were selected to be the hybrid parents. “Wan Yingui” is a 25 years old male cultivar. “Huangchuan Jingui” is a 20 years old hermaphrodite cultivar and was employed as female parent. So the bagging and emasculation are necessary in the process of artificial pollination. The parents showed obvious differences in gender, corolla color and leaf traits. The hybridization was performed at the Garden of Huangchuan Jingui , Huangchuan County, Henan Province, in the fall of 2014, and the fruits were harvested in the spring of 2015. The seeds would usually take 1–2 years to germinate normally. To shorten this period, the hybrid seeds were subjected to in vitro embryo culturing according to Wangjun et al.  at the GGenomic DNA samples were evaluated using 1% agarose gels and an ND-1000 spectrophotometer  to ensure it suitable for SLAF-seq in terms of integrity and quality.RsaI, ATP, T4 DNA ligase, and Duplex Tag-labeled Sequencing adapters at 37°C. Then the digestion-ligation DNA was used for the subsequent polymerase chain reaction (PCR), together with the essential components of dNTP, Taq DNA polymerase, and primers. The products were then purified, pooled and separated via electrophoresis on 2% agarose gel. The fragments between 314 and 414 bps were extracted and sequenced using the Illumina HiSeq 2500 system  at the Biomarker Technologies Corporation in Beijing.The SLAF library construction and high-throughput sequencing were conducted as the report of Sun et al.  accompanSLAF marker identification and genotyping were accomplished as following. Low-quality reads in each cycle  were throw away by real-time monitoring during sequencing. Then, those with clear index information were mixed together in view of their sequence similarity, which can be ascertained by BLAT Kent, . The seq2) was carried out to examine the Mendelian segregation ratio for each marker. Markers at the P < 0.05 significance level were initially excluded from the core linkage map and separately inserted as accessory markers at a later stage. Map construction was achieved according to the method of HighMap, which was detailed by Liu et al.  content was 37.5% on average. Subsequently, 173,537 SLAF markers were derived from the high quality reads, of which 135,540 and 130,698, respectively, were exploited from the female and male parents. The read numbers for SLAFs were 5,575,444 in the female parent and 5,828,967 in the male parent, with the sequencing depth of each SLAF was 43.01- and 42.66-fold, respectively. In the 129 progenies, the reads numbers for the SLAFs varied from 556,112 to 1,592,274, and the development of SLAFs ranged from 100,910 to 122,711 with a marker depth of 5.27-fold on average for each individual  according to the modified LOD (MLOD) scores between markers and the linkage analysis. The percentage of SLAF markers available for mapping was about 92.64%, with 9,175 SLAFs available for the female map, 7,196 SLAFs for the male map, and 14,189 SLAFs for the final integrated map  were also on LG7. The others were scattered on 14 LGs with SDR numbers ranging from 1 to 5.The basic characteristics of the markers for the final map are shown in Table The 14,189 SLAFs on the integrated map were composed of three types of markers. These were 11,228 “SNP-only,” 559 “InDel-only,” and 2,402 “SNP & InDel” types, with percentages of 79.13, 3.94, and 16.93%, respectively. The marker distribution of the three types on the 23 LGs are shown in Figure Heat map and haplotype map were used to estimate the map quality obtained in this work. The genetic map was based on multi-point recombination analysis. The closer the markers were, the smaller the recombination rate was. The heat map showed the recombination relationship between markers Figure . The hapO. fragrans, a species for which there is relatively little genomic information. This could be a useful starting point for further genetic studies on this plant.Genetic linkage maps are useful tools for genomic studies. They can elucidate the genome structure and facilitate the identification of molecular markers pertinent to biological traits in an experimental segregating progeny. It is particularly important to construct maps for long generation species, such as tree species. This study has developed the first reference genetic linkage map for O. fragrans, such as Double Haploid (DH), Recombination Inbred Lines (RILs) and F2 progeny, which have been widely used to construct the maps for many different crops. However, O. fragrans is a highly heterozygous species where the F1 hybrids often show substantial and different types of segregation   with the size of chromosomes reported by Li et al. . The similar positive correlation was also detected in female and male maps , so the different number of markers on the LGs would cause the difference length of the same LGs in both parent maps. LG8 showed the most difference in length and the order of some markers between the male and female maps, which may be caused because the relative positions of tightly linked markers are usually uncertain due to the effects of missing values and to differences in segregation information among markers  were added to the final map, which should improve any future QTL genotype mapping.Segregation distortion is a ubiquitous phenomenon in many species, which skews the frequency of alleles from the expected Mendelian ratio (Faris et al., O. fragrans. No phenotypic traits were located on the present map because the progenies in our study were still seedlings, and no traits of ornamental or agronomic interest could be segregated and evaluated because of the juvenile nature of the seedlings. The mapping population resources have been planted at the Garden of Huangchuan Jingui for several months where the climate is suitable for the growth of this species. Once the progenies become sexually mature, this high-density linkage map could offer a useful platform for future QTL mapping, including the important traits of flowering time, corolla color, flower fragrance, and cold resistance. The markers on this map were developed at level of the whole genome. Therefore, the constructed map also serves as an important reference for gene cloning and genome structure investigations into O. fragrans.It is the first reference map that was reported in this study for FS and WY designed and organized the entire project. YxH, WY, MD, and YjH performed the experiments. YxH analyzed the data and drafted the manuscript, and FS revised it. 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. The reviewer PG and handling Editor declared their shared affiliation


Many mortality risk scoring tools exist among patients with ST-elevation Myocardial Infarction (STEMI). A risk stratification model that evaluates STEMI prognosis more simply and rapidly is preferred in clinical practice.We developed a simple stratification model for blood examination by using the STEMI data of AMI-Kyoto registry in the derivation set  and assessed its utility for mortality prediction in the validation set (n = 521). We selected five variables that significantly worsen in-hospital mortality: white blood cell count, hemoglobin, C-reactive protein, creatinine, and blood sugar levels at >10,000/μL, <10 g/dL, >1.0 mg/dL, >1.0 mg/dL, and >200 mg/dL, respectively. In the derivation set, each of the five variables significantly worsened in-hospital mortality (p < 0.01). We developed the risk stratification model by combining laboratory variables that were scored based on each beta coefficient obtained using multivariate analysis and divided three laboratory groups. We also found a significant trend in the in-hospital mortality rate for three laboratory groups. Therefore, we assessed the utility of this model in the validation set. The prognostic discriminatory capacity of our laboratory stratification model was comparable to that of the full multivariable model . In addition, we divided all cases  into three thrombolysis in myocardial infarction (TIMI) risk index groups based on an In TIME II substudy; the cases were further subdivided based on this laboratory model. The high laboratory group had significantly high in-hospital mortality rate in each TIMI risk index group .This laboratory stratification model can predict in-hospital mortality of STEMI simply and rapidly and might be useful for predicting in-hospital mortality of STEMI by further subdividing the TIMI risk index. Acute myocardial infarction (AMI) is a disease with poor in-hospital prognosis worldwide. ST-Elevation Myocardial Infarction (STEMI) has a worse prognosis. The development of primary percutaneous catheter intervention (PCI) succeeded in decreasing the in-hospital AMI mortality rate . NonetheSeveral risk stratification models have been reported. The thrombolysis in myocardial infarction (TIMI) risk score could accurately predict STEMI outcomes . The TIMTherefore, we developed and assessed a simple and easy risk stratification model from the combination of blood examination variables for in-hospital mortality rate prediction of STEMI.The AMI-Kyoto multicenter risk study is a prospective, multicenter observational study of patients with AMI who were transferred to 15 participating institutions –7. A totThe patients’ demographic information, cardiovascular history, and risk factors  were recorded. Hyperlipidemia was defined as a total cholesterol level of ≥220mg/dL or the use of cholesterol-lowering agents; hypertension was defined as SBP of ≥140/90 mm Hg or the use of antihypertensive treatment; and diabetes mellitus was defined as a fasting blood sugar (BS) level of ≥126 mg/dL or the use of specific treatment. The SBP at admission was defined as the first SBP recorded in the supine or sitting position upon arrival at the emergency room or out-patient clinic. Based on the WHO criteria, anemia was defined as hemoglobin (Hb) level of <13.0 g/dL and <12 g/dL for men and women, respectively. All in-hospital data were transmitted to the center at the Department of Cardiovascular Medicine in Kyoto Prefectural University School of Medicine for analysis. The study protocol was approved by each hospital’s ethics committee. Because this study corresponds to an observational study to use only existing data from medical records, we performed the verbal informed consent without the agreement document. We indicated record of the informed consent to the electronic medical recoding system. After each institution approved the participation in this study, this study was overall approved by the ethics committee of Kyoto Prefectural University of Medicine as a generalization organization. In addition, this study was approved by the each institutional ethics committee participated in this study .Emergency coronary angiography (CAG) was performed using the standard technique. The coronary flow in the infarct-related artery was graded based on the classification used in the TIMI trial. Coronary stenosis was evaluated based on the American Heart Association classification, and significant coronary artery stenosis was defined as a reduction of at least 75% of the internal diameter in the right and left anterior descending or left circumflex coronary arteries and their major branches, or 50% of the internal diameter in the left main trunk. Multi-vessels as culprit arteries were defined as simultaneous thrombosis of multiple coronary arteries or undetermined culprit artery in the presence of multiple stenotic vessels on the initial CAG. Primary PCI was performed according to the standard techniques, using the appropriate strategy, based on the discretion of the attending physician.Anemia, renal failure, hyperglycemia, increased C-reactive protein (CRP) levels, and/or white blood cell (WBC) count were reported as in-hospital prognosis factors for AMI –14. Baset-tests for discrete and continuous variables, respectively. To define the cutoff values, we assessed the relationship between each of the five factors with in-hospital mortality rate and plotted them by using the spline curve. To avoid the complex modeling, the spline curve was taken as the range between 0 and 30,000 for WBC, between 6 and 20 for Hb, between 0 and 10 for CRP, between 0 and 5 for Cr, and between 0 and 500 for BS , respectively. The derivation and validation sets were compared by using the chi-square and unpaired Student’s 0 for BS . The oddThe mean age of the patients in the derivation and validation sets was 70.0±12.4 and 70.1±12.9 years, respectively. No significant differences were found in age, sex, cardiovascular risk factors, and family history. Although most patients were transferred to the hospital by ambulance, more than one fourth of patients arrived by self-triage. SBP and HR upon arrival were identified as prognostic factors in the TIMI risk index, which were similar between the derivation and validation sets. Killip classification IV was observed in 9.7% and 12.8% patients in the derivation and validation sets, respectively. No significant differences were found in the severity between the two sets; peak CPK levels were 1,884 and 1,951 mg/dL for the derivation and validation sets, respectively. The laboratory data related to STEMI mortality rate were reported to be similar in the derivation and validation sets . SimilarPrimary PCI was performed in nearly all cases (>95%) for STEMI patients. Infarct-related coronary artery and multi-vessel disease were similar between the two sets. We found that approximately 60% of cases had TIMI 0 flow in the two sets. TIMI classification and the use of supporting devices also showed no significant differences between the two sets. Moreover, similar population distribution was found between the two sets.WBC, anemia, CRP, renal function, and hyperglycemia have been reported as independent prognostic factors for AMI. We assessed the predictive power of these laboratory variables for the in-hospital STEMI mortality rate in the derivation set. Each variable  was also associated with a significantly high in-hospital mortality rate in the derivation set . Based oTo assess and triage the in-hospital mortality for STEMI simply and rapidly, we developed a risk stratification model from the combination of laboratory variables. We decided to assign points to each factor based on the beta coefficient of WBC and created a model that calculates the total points in the derivation set. Hb < 10 g/dL and CRP > 1.0 mg/dL are assigned 1 point because of the approximate beta coefficient of WBC > 10,000 /μL; Cr > 1.0 mg/dL is assigned 3 points because of the three-fold beta coefficient of WBC > 10,000 /μL; and BS > 200 mg/dL is assigned 2 points because of the two-fold beta coefficient of WBC > 10,000/μL . A signi3. We divided the TIMI risk index into three groups based on the mortality rate in the InTIME II substudy. Cases with less than 22.5, from 22.5 to less than 30, and 30 or more points were classified as low, moderate, and high TIMI risk index groups, respectively. We created ROC curves for our laboratory model and TIMI risk index in all cases. The prognostic discriminatory capacity of our laboratory model was comparable to that of the full multivariable model . This result shows that the performance of our laboratory model is comparable to that of the TIMI risk index  by using the three values reported as predictors of STEMI in an InTIME II substudy sk index . The thrsk index .The results of this registry showed that each of the five factors  in the blood examination worsened in-hospital STEMI prognosis. In addition, the combination of these five factors could also predict the in-hospital mortality of STEMI. This laboratory risk stratification model combined with the TIMI risk index could be helpful in predicting the in-hospital STEMI prognosis in detail.Few studies assessed the combination of laboratory variables for AMI prognosis. Patients with chronic kidney disease (CKD) were reported to have a significantly higher in-hospital mortality rate than the patients without CKD. Renal failure is also reported to be a cause for cardiovascular events, such as heart failure due to left ventricular insufficiency , 15–17. In this study, each of the five factors was also verified to be significant in-hospital prognostic variables of STEMI. Combination of these laboratory variables could also be a risk model to predict the in-hospital STEMI prognosis. Several risk stratification models have been reported in the literature. The TIMI risk index is defined by age, SBP, and HR . This riThis study has several study limitations. First, this study is an observational analysis of a relatively small number of patients. Second, data regarding clinical background characteristics and angiographic results of primary PCI were unavailable for all study participants. We did not have appropriate detailed data regarding left ventricular function, ischemic time, and in-hospital medical treatments, which might be predictors for the in-hospital mortality of STEMI. In addition, the cutoff values for each of the five variables were determined by prioritizing figures within the spline curve created using the hospital prognosis with figures that can be used easily and conveniently in clinical practice. We could not obtain the data regarding the background wherein primary PCI was not performed. However, more than 90% of the subjects underwent primary PCI, and more than 90% were able to obtain TIMI III grade coronary flow as the TIMI classification after primary PCI, which is similar to actual clinical conditions. Despite these limitations, this laboratory risk stratification model could be useful for the evaluation of the STEMI prognosis.This laboratory stratification model could predict in-hospital mortality of STEMI simply and rapidly. This model could be useful for the detailed prediction of in-hospital STEMI mortality rate by further subdividing the TIMI risk index.S1 FigWBC, White blood cell; Hb, Hemoglobin; CRP, C-reactive protein; Cr, Creatinine; BS, Blood sugar level.(TIF)Click here for additional data file.S2 FigWBC, White blood cell; Hb, Hemoglobin; CRP, C-reactive protein; Cr, Creatinine; BS, Blood sugar level.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Life expectancy at birth (LE) is a comprehensive measure that accounts for age-specific death rates in a population. Shanghai has ranked first in LE in China mainland for decades. Understanding the reasons behind its sustained gain in LE provides a good reflection of many other cities in China. The aim of this study is intended to explore temporal trend in age- and cause-specific gains in LE in Shanghai and the probable reasons lay behind.Joinpoint regression was applied to evaluate temporal trend in LE and the long time span was then divided accordingly. Contributions to change in LE (1973–2015) were decomposed by age and cause at corresponding periods.LE in Shanghai could be divided into four phases ie., descent (1973–1976), recovery (1976–1998), rapid rise (1998–2004) and slow rise (2004–2015). The growing LE was mainly attributed to reductions in mortality from the elderly populations and chronic diseases such as cerebrovascular disease, chronic lower respiratory disease, and gastrointestinal cancers .The four-decade sustained gain in LE in Shanghai is due to the reductions in mortality from the elderly and chronic diseases such as cerebrovascular disease, chronic lower respiratory disease, and gastrointestinal cancers. Further growth momentum still comes from the elderly population.The online version of this article (10.1186/s12889-018-5112-7) contains supplementary material, which is available to authorized users. Life expectancy at birth (LE) is a comprehensive measure reflecting health conditions of a certain country (or region). LE equals the average years a person is expected to live based on the overall mortality level of a population, hence, is a type of demographic data most commonly used to portray the social and health conditions, as well as well-being of a society. , 2.In October 2016, the Chinese government implemented “healthy China 2030”, a program of action to promote a healthier China in the next 15 years. Program aims include incorporating health into all policies, ensuring individual’s health in an all-round and full cycle, and improving health and health equity. An additional goal is to increase the national LE to 79 by 2030 [China has made extraordinary progress on LE since its founding in 1949. LE has more than doubled, from 35 years before 1949, to 76.3 in 2015 . LE in SShanghai has taken lead in LE for 12 years nationwide . TemporaSince comprehensive Mortality Registration System in Shanghai dates back to 1973 , our stuAlthough the 42-year information on detailed causes of death for Shanghai as a whole was unavailable, LE and individual mortality data were collected from Pudong New Area instead. Pudong New Area accounts for one-fifth of the total population in Shanghai, consisting of both urbanized and rural places . Pudong The Mortality Registration System of Pudong New Area, covers medical institutions of all levels and data is checked against local population registry on a monthly basis. Periodic evaluations, data cleaning and compilation have been done at both the county and provincial levels according to standard guidelines . The infAs the data covered a long time span, the 42-year data was divided into three parts according to coding methods. Data for 1992–2001 and 2002–2015 were coded based on the International Classification of Diseases, 9th and 10th Revision (ICD-9 and ICD-10), respectively. Historical records before 1992 had been in paper form and were digitized and recoded based on ICD-10. The recoded historical data was checked against historical annual reports by cause and age. In case of a difference in rules for selecting underlying cause of death (UCOD), UCOD was modified according to ICD-10 to avoid inconsistency in classification. All the data was then analyzed according to the Chinese Classification of Diseases (CCD), due to previous use in Shanghai’s annual report and assisted to bridge ICD-9 and ICD-10.All causes of death were coded by rigorously trained clinicians, and each record was further verified by local Center for Disease Control and Prevention (CDC). Review of medical records, reports from family members, or police records were carried out if there were any discrepancies. Ill-defined data was greatly reduced through above- mentioned methods.Joinpoint regressions have been widely conducted to explore potential changes in trends , 18. TheTherefore, the study introduced joinpoint regression to scientifically evaluate a temporal trend in LE during the 42 year period. The method identifies whether there are statistically significant differences in the trend of LE and quantifies the speed with which the increase or decrease occurs. The year was assigned as an independent variable while the annual LE is a dependent variable. The maximum number of joinpoints was set at 5 and Heteroscedastic Errors Option was set at constant variance. The Grid Search method was selected and Monte Carlo simulation was performed with the number of permutations set to 4499 to determine the optimal number of change-points in segmented line regression.LE was estimated on the basis of annual abridged life tables, which were constructed with reference to a standard table . The ageGiven the potential variation of data quality, the quality of data was evaluated across different years by a recommended method . The metStatistical analysis was performed with Stata/SE 11.0 . Joinpoint regression analyses were carried out using Joinpoint Regression Program, Version 4.0.4 .Between 1973 and 2015, LE increased from 74.1 to 83.2 and this trend could be divided into four phases. The descent (1973–1976) saw LE dropped dramatically from 74.1 to 72.0 ). LE stepped into a recovery phase (1976–1998), increasing steadily from 72.0 to 76.7 ). A sharp increase was observed during 1998–2004 ). After then, trend in LE turned into a slower increase and reached to 83.2 in 2015 ) , cardiovascular diseases , cancer , perinatal deaths , external causes  and digestive system diseases .At the recovery phase, increases in LE mainly resulted from respiratory system diseases , external causes , digestive system diseases , cardiovascular diseases , infectious diseases , and cancer . The six disease groups accounted for over 76% of the total increase in LE.For the rapid rise, cardiovascular diseases outreached respiratory system diseases and became the leading contribution to the increase in LE . Followed by cancer , congenital malformations , perinatal deaths  and external causes . The six disease groups accounted for over 93% of the total increase in LE.For the slow rise, increases in LE mainly resulted from cancer , external causes , respiratory system diseases , cardiovascular diseases , infectious diseases  and digestive system diseases . The six disease groups accounted for over 77% of the total increase in LE , cancer , chronic rheumatic heart disease, and neonatal asphyxia and trauma.Chronic lower respiratory disease took the lead in contribution to increase in LE at recovery phase, followed by respiratory tuberculosis, cirrhosis, self-harm, pneumonia, cancer , and cardiovascular diseases (pulmonary heart disease and chronic rheumatic heart disease). These ten causes accounted for 63.6% of the total increase at that time.For the rapid rise, cerebrovascular disease overtook chronic lower respiratory disease and became the leading cause of contribution. Neonatal asphyxia and trauma, ischemic heart disease, congenital heart anomalies, cancer , poisonings, and hepatitis also made great contributions. As a result, up to 82.0% of the total increase was attributed to these diseases.For the slow rise, the two leading causes remained same, ie., cerebrovascular disease and chronic lower respiratory disease. In addition, contributions of LE from the three leading cancer  improved. External causes  also made contributions, accounting for over 11% of increase in LE. All the ten causes contributed to 74.7% of the total increase in LE . Cardiovascular diseases, represented by cerebrovascular disease, led the contribution to LE in Shanghai recent years . Since pHelicobacter pylori and other parasites (schistosome) infections were potential causes for the reduction of the incidence [Consistent with findings in developed countries, our data showed a strong and continuous contribution from declining mortality of stomach, esophageal, and liver cancer . Improvencidence –42.Lung cancer contributed little, given the fact that it ranked first in mortality in cancer. Moreover, colorectal cancer and pancreatic cancer showed increase tendencies of mortality , 44. CanAs a microcosm of China, Shanghai also witnessed the huge impact of political and social factors on LE. LE set back by over 2 years during 1973 to 1976. A majority of disease groups made negative contribution to LE. Among them, external causes represented by self-harm is particularly prominent. Drowning and self-harm were the top five negative contributors to LE during the descent phase, but self-harm turned to be the fourth positive contributors to LE at the recovery phase. The Cultural Revolution in China (1966–1976) left a scar in Chinese history. Social unrest, distorted moral values, and repressed emotion led to extremes. As a result, the contribution of self-harm on LE differed greatly during the descent and recovery phase. Changes of political and social environments strongly influence LE, as proven in other countries , 46. A p8 However, Shanghai did not rank first in GDP per capita among the provinces in the first group in China. Reasons for its high LE is likely due to its long-term emphasis on public health. Shanghai is one of the earliest and best provinces to implement the strategy of modern TB (tuberculosis) control in China [The top 10 disease groups of death for China and Shanghai were almost same in rank and type  study . CCD is In addition, the population was analyzed as a whole in this study without distinction between male and female. In our view, the overall impact on human from achievements in medicine and public health, plus the political and social factors were approximately same across both sexes. However, we admitted that there were gender differences, especially in specific diseases involved. We will explore this issue in greater detail in a future article . Reduction in infant mortality also plays an important role, especially in the recovery and rapid rise phase; however, its impact dropped sharply in the slow rise phase. Coronary heart diseases and lung cancer showed less contribution to LE, compared with cerebrovascular disease and gastrointestinal cancers. Political and social factors are critical for analysis as well. LE in Shanghai set back by over 2 years during 1973 to 1976, particularly due to external causes represented by self-harm. Understanding the LE of the forerunner will have reference value for many other cities in China. Age- and cause- specific decomposition of LE assist in determining key population and diseases, set specific goal for further achievement in LE, and ensure realization of “Healthy China 2030”.In conclusion, the four-decade sustained gain in LE in Shanghai is attributed to the reductions in mortality from the elderly population and chronic diseases, such as cerebrovascular disease, chronic lower respiratory disease, and gastrointestinal cancers. Further growth momentum in LE continues to stem from the elderly population.Additional file 1: Table A1.Table A2. Top 10 Causes of death of the nation, province and study area in 2015. Table A3. The sensitivity analysis of Arriaga’s decomposition method. Table A4 The characteristics of the population at different time. (DOCX 29 kb)List of causes of death based on CCD (Chinese Classification of Diseases) and the corresponding ICD-10 codes. Additional file 2: Figure S1 A1.Percentage of all deaths coded to garbage codes by age in different years. (TIFF 1817 kb)


This response refers to:Int. J. Mol. Sci. 2016, 17, 1042.Xu, Z.; Liu, Y.; Zhou, S.; Fu, Y.; Li, C. Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques. Int. J. Mol. Sci. 2016, 17, 1916.Merlot, A.M.; Sahni, S.; Lane, D.J.R.; Richardson, V.; Huang, M.L.H.; Kalinowski, D.S.; Richardson, D.R. Letter to the Editor: Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques and.  International Journal of Molecular Sciences. We are aware of the fact that Dr. Richardson in a leading researcher in the field and both respect Dr. Richardson and regularly refer to the many publications from his group.Thank you for forwarding the concerns (or comments) from Dr. Richardson  regardinNonetheless, we would like to respond to the specific concerns raised by Dr. Richardson.In reading the article by Xu, Z., et al. entitled “Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques” , there is evidence that some of the key methods and comparisons utilized in this study are non-optimal and problematic.We would also like to bring to the attention of readers a number of important flaws and factually incorrect statements in this article.The paper examines the binding of the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), to human serum albumin (HSA) and DNA and states  that “no studies have examined the effects of the interaction between Dp44mT and biological molecules, such as proteins and nucleic acids”. However, two studies from our group have previously comprehensively examined the binding of Dp44mT to HSA  and DNA . These previous studies performed very similar experiments, including cytotoxicity studies, circular dichroism (CD), fluorescence and molecular docking studies, and hence, the current paper appears as a duplication and cannot be considered a novel contribution to the literature. Significantly, the authors knew of one of these articles as they directly cite it (REF #13 in their manuscript). Such statements appear misleading and inappropriate and this requires correction.Dr. Richardson’s concerns regarding the sentence “no studies have examined the effects of the interaction between Dp44mT and biological molecules, such as proteins and nucleic acids” in the introduction section are justified and we do realize that the sentence is equivocal. Unfortunately, this sentence was changed as a consequence of language editing of the original manuscript. We are sorry for our carelessness, but we did not have the intention to mislead readers. Consequently, the sentence should be changed back to the original version: “information related to the effects of the interaction between Dp44mT and biological molecules such as human serum albumin (HSA) or DNA has not yet been fully and systematically studied”.Secondly Dr. Richardson considers our work a duplication and not a novel contribution to the literature. We did read Dr. Richardson’s papers ,4. HowevMoreover, the following methods, comparisons and interpretations of results may be unreliable and should be taken into consideration when interpreting the results presented:-Characterization and purity of Dp44mT:The authors synthesized Dp44mT in their own laboratory and present only the mass spectrum to confirm its structural identity. Notably, other methods of structural characterization  and chemical purity  are required to ensure that the Dp44mT was indeed correctly synthesized, and was of the high purity necessary for biological studies. As such, the purity of the compound utilized is unclear.1H-NMR, 13C-NMR, elemental analysis, and HPLC used for structural characterization and purity assessment, ensuring Dp44mT was synthesized with high quality for a biological study. Dp44mT was synthesized by reaction of commercial 2-dipyridylketone (Sigma) with N,N-dimethyl-3-thiosemicarbazide (Sigma). This is a simple one-step condensation reaction, we finished the reaction, and following specific recrystallization, a high purity of Dp44mT was achieved. Purity assessment was performed and in accordance with your suggestion, the requested information is now included in the correction . Alternative techniques should have been used, such as viable cell counts. Hence, the results obtained cannot be considered as reliable.The MTT-based method is still widely used to assess a drug’s cytotoxicity or proliferation inhibition ,7,8. Giv-Comparison of HepG2 proliferation studies to previous literature:The effect of HSA on the anti-proliferative activity of Dp44mT was performed using HepG2 cells in this article. Notably, the authors then compare the results obtained in this cell line to previous studies  and state : “growth inhibition induced by Dp44mT in the presence of equimolar HSA was similar to that without HSA, in contrast to previous findings .” Significantly, the referenced study examined the effect of HSA and Dp44mT on the proliferation of SK-N-MC cells , not HepG2 cells, and thus, cannot be directly compared.To address the phenomenon of the effect of HSA on drug cytotoxicity, we qualitatively compared our results with the reference’s findings , even th-Circular dichroism method—use of DMSO:DMSO has high far-UV absorbance and should not be used in CD measurements . The implementation of DMSO leads to high tension (HT) voltage values and the saturation of the detector , leading to erroneous results. Alternative solvents that do not interfere with CD spectra, including ethanol and acetonitrile, should be used to dissolve the ligand for subsequent addition to the protein.We do agree with Dr. Richardson’s point of view: DMSO potentially affects the CD spectrum. In our experiments, DMSO was used at very low concentration, and the CD spectra were corrected for its contribution. Hence, the data obtained was objective. Future related CD experiments may use ethanol or acetonitrile if the sample under investigation is soluble in those solvents.-Circular dichroism interpretation:Notably, upon examination of their experimental CD results, the authors state : “In the presence of different concentrations of Dp44mT, a slight reduction in negative ellipticity was observed, indicating that the α-helix content of the protein was decreased . The contents of secondary structure can be calculated based on experimental data , but it was limited due to larger errors.” However, the nature of the large error is not well explained. Unfortunately, the authors rely on simulated data to assess the effect of Dp44mT on the secondary structure of HSA. Critically, the simulated secondary structural data for HSA alone reported by the authors does not reflect the known levels of α-helical or β-sheet content of HSA previously published . Hence, again, there are serious uncertainties regarding these data.The contents of the secondary structure can be determined either via direct calculation (based on equations) or, as another option, through simulation-based on experimental data. Because DMSO was used in our CD experiment, the contribution of DMSO to the CD spectra required correction of the spectra. Due to direct calculation for the α-helix content based on a MRE value at 208 nm, after correction for DMSO, the averaged CD curve at given wavelength (208 nm) may be less accurate. Therefore, simulation of the curve may decrease the error upon direct calculation, and consequently we used the simulation program (K2D2) rather than the analytical equation, which is also widely used in CD spectrum simulation ,10,11.


The research has several implications for treatment, clinical health psychology, family support and education.The current study investigated attachment representations and complex trauma in a sample of gender dysphoric adults. Although it has been proven that the psychological wellbeing of gender diverse persons is largely mediated by family acceptance and support, research on their relationships with parental figures is scarce. A total of 95 adults took part in the study. The attachment distribution was as follows: 27% secure, 27% insecure and 46% disorganized. Regarding early traumas, 56% experienced four or more traumatic forms. Further, gender dysphoric adults showed significantly higher levels of attachment disorganization and polyvictimisation, relative to controls. Comparisons of subgroups, defined by natal gender, showed that  DSM-5  is described in the DSM-5  as a conDSM-5 . De Cuypes e.g., , which aes e.g., . With rees e.g., . Rather,es e.g., . The onses e.g., . Many ches e.g., , 2013. Wes e.g., . Whetheres e.g.,  and poores e.g., , and thees e.g., . Furtheres e.g., . Howeveres e.g., . As Ristes e.g.,  and thuses e.g., . Howeveres e.g., , but fewtrans adults with early onset GD (first childhood)  , in ordeildhood) , which iildhood) , in addiComplex trauma can be understood as a set of experiences of cumulative, chronic and prolonged traumatic events, most often of an interpersonal nature, involving primary caregivers and frequently arising in early childhood or adolescence . MaltreaThe relationship between relational trauma and GD has often been described as univocal. Many authors e.g.,  have spetrans women, reported that the majority of the women had failed to disclose their gender incongruence to parents and siblings in early adolescence, and thus had been unable to benefit from the protection of family support, which has been shown to be a protective factor for mental health  with transsexual or gender-variant populations. trans people and may interfere with their gender expression. Specifically, we aimed at exploring gender variant persons’ attachment representations and history with primary figures in early experiences of trauma and loving caregiving, and comparing these findings with those of a cisgender control group. In order to explore the internalized aspects of parental relationships in our subjects, we narrowed our analysis to the Adult Attachment Interview subscales concerning states of mind directly connected to parental figures . At a deeper level of enquiry, we then performed comparisons of subgroups. To analyze attachment and trauma, we broke our sample down according to gender (trans women vs. trans men). Additionally, regarding trauma, we compared trans women to male controls and trans men to female controls, in order to explore the differences in natal males and females between transgender and cisgender developmental paths. At the heart of our research was an intention to update psychological and clinical perspectives on the developmental environment of trans persons, highlighting the impact that adversities in the family context may have on their adult adaptation. Our aim was to read these adversities not as they have been read in the past – as etiological factors for GD – but as instances of risk that should be carefully managed by psychologists working with trans people. Ultimately, the purpose of this study was to contribute to and facilitate trans persons’ development of a free and personal identity during their transition path.In response to the identified gaps in the literature, this study aimed at gaining in-depth knowledge of some problematic features that may be grounds for psychological complications in To sum up, we aimed to test the following hypotheses:(1)The first hypothesis postulated that in our sample of gender dysphoric people unsecure and disorganized attachment classifications were more frequent, relative to controls;(2)The second hypothesis assumed that in the gender dysphoric sample higher frequencies of traumatic experiences could be found within early caregiving relationships, compared to controls;(3)The third hypothesis postulated that in the gender dysphoric sample early experiences of a loving caregiving were less frequent compared to controls;(4)trans women and trans men.The forth hypothesis assumed that it was possible to find differences regarding attachment and complex trauma between trans women, 21 trans men). The sample was recruited from the Service for Adaptation of Physical Identity to Psychological Identity Clinical Center  and the Gender Dysphoria Psychological Service – Department of Clinical Neurosciences, Reproductive and Odontostomatological Sciences . All participants declared an early onset of GD (first childhood). The mean age of the full sample was 29 years (SD = 7.2); the same mean age was identified for trans women (SD = 7.8) and trans men (SD = 5.0). All trans men were Italian, while 86% of trans women were Italian and 14% were South American. With respect to the level of education, 3% had an elementary license, 39% had a middle school license, 51% had a high school diploma and 7% had a college degree.The study was conducted with 95 GD adults (74 SD = 10.2); the mean age of males was 35 years (SD = 11.9) and the mean age of females was 33 years (SD = 5.6). Aside from 2% of the sample, all were Italian. With respect to the level of education, 27% had a middle school license, 54% had a high school diploma and 19% had a college degree. The overall sample was recruited expressively asking for a participation secured through a written informed consent procedure that required active consent from participants. The current study has been attended within the Ph.D. context at the Department of Dynamic and Clinical Psychology  and received the approval from the Ethical Committee of the Department (n° 24/2016).The control group, which was recruited through snowball sampling by the authors in Rome and Naples, was composed of 123 non-clinical persons . The mean age of the total sample was 34 years  and instances of threat or abuse. Following this, questions are posed concerning respondents’ early reactions to separation from a caregiver and significant loss. The interview transcripts are scored on thirteen 9-point scales. Five scales assess the “probable childhood experience” with caregiving figures , whereas eight scales assess the current mental state with regard to attachment . The AAI score is based on the participant’s ability to produce coherent narratives of childhood experiences with caregivers that classify the respondent as Secure/Autonomous (F), Dismissing (Ds), Entangled/Preoccupied with respect to attachment (E) or “Cannot Classify” (CC) when a global breakdown in the organization of discourse arises. An interviewee may also be assigned an Unresolved/Disorganized state of mind (U) concerning past abuse or loss in association with a best-fitting primary classification. Psychometric testing and meta-analyses have provided evidence of the AAI’s stability and discriminant and predictive validity in both clinical and non-clinical populations . In the The ComplexTQ is a 70-item scale for the retrospective assessment of multi-type maltreatment, measuring lack of care , abuse  and other traumatic experiences, such as rejection, role reversal, exposure to domestic violence, separation and loss. The instrument is available in two different versions: clinician-report and self-report. The questionnaire assesses adverse experiences from childhood (to the age of 14) involving maternal, paternal and other attachment figures, separately. In the current study, the clinician-report version was used and applied to the AAI transcripts. The questionnaire requires approximately 15 to 20 min to complete, depending on the length of the interview transcript. Scores for the presence and frequency of traumatic experiences in each domain are automatically provided by the software.t-test comparisons, we performed a multiple testing correction using the Benjamini–Hochberg (BH) method .Concerning the analyses of the current study, for the attribution of attachment categories we used the scoring system of ) method  to adjussr = 3.4) and U/CC (sr = 2.7) classifications.Analysis of the 4-way distribution of AAI transcripts highlighted that, within the control group, 61% were classified as F, 19% were classified as Ds, 7% were classified as E and 13% were classified as U. With regard to the GD participants, 27% were classified as F, 14% were classified as Ds, 13% were classified as E and 46% were classified as U. Focusing on the disorganized portion of the GD sample, 19% were classified as U, 17% were classified as CC and 10% were classified as both U and CC. Despite the heterogeneity of the GD persons’ disorganized profiles, analysis of the standardized residuals highlighted an overrepresentation of CC , there was an underrepresentation of F classifications (sr = -2.7) and an overrepresentation of insecure categories (sr = 2.5). Regarding the distribution of organized  and disorganized (U and CC) classifications , in the GD sample, disorganized states of mind were overrepresented (sr = 3.5) and, to a lesser extent, fewer organized attachments (sr = -2.2) than expected were found.Table 2). Analyses of the 4-way distribution showed that our GD group differed significantly from normative samples – both international  and Italian , and, to a lesser extent, to the Italian clinical sample  – whereas no difference was found between our GD group and the international clinical sample. As to the comparison of organized and disorganized AAI classifications, the GD group was strongly significantly different from normative samples – both international  and Italian  – although it showed no differences relative to clinical samples.We then compared the GD group to normative and combined clinical samples from international  and natiTable 3 shows the mean scores of the three scales for the two groups. In GD patients, after the BH correction, only Anger toward fathers resulted significantly higher, relative to the control group.As mentioned above, we opted to explore the AAI state of mind scales for Idealization, Derogation and Anger. trans women and trans men in the GD sample, the 4-way AAI distribution, the distribution of secure vs. insecure AAI classifications and the distribution of organized vs. disorganized AAI classifications showed no significant differences. Concerning the AAI state of mind scales, the only significant difference between the two subgroups was observed with regard to Idealization , showing that trans women idealized their mothers to a greater extent.With regard to the comparison between Presence of a traumatic history was evaluated with the ComplexTQ, which considered participants’ experiences of many forms of relational trauma to the age of 14, within primary relationships. Results showed that 10% of GD participants had not experienced any early adversity, 13% had experienced one form of trauma, 8% had experienced two forms, 13% had experienced three forms and 56% had experienced four or more forms. In the control group, 30% of participants had not experienced any form of trauma, 37% had experienced one form of trauma, 16% had experienced two forms, 9% had experienced three forms and 7% had experienced four or more forms.Table 4 shows the distribution of individuals (GD patients and controls) with experience of fewer than four traumatic forms against those who were polyvictimised, showing experience of four or more forms of trauma. We opted for this grouping, following the examples of 2 = 61.881, p = 0.000, effect size φ = 4.19], showing a robust overrepresentation of polyvictimisation (sr = 5.0) and an underrepresentation of fewer than four experienced forms of trauma (sr = -3.2).We also compared our GD group with the clinical sample of For the comparison of our sample and controls regarding the frequency of experiences of various forms of trauma, the reader is referred to the following subsection, where the comparison is broken down according to gender.Table 5. In our sample, also after the BH correction, significantly lower scores were found on the following scales, all regarding the relationship with the mother: Neglect and Reject, and, to a lesser extent, Psychological Abuse, Physical Abuse and Witnessing Domestic Violence. There were no significant differences between the two samples for the Loss scales.The comparison with literature data  concernitrans women and male controls, and subsequently between trans men and female controls.We first compared trauma frequency between Tables B. Regarding the comparison between trans women and male controls, trans women resulted to have, following the BH correction, significantly higher scores on the following scales: father’s Reject, Physical and Psychological Abuse, and Neglect from both parents. Fathers’ Neglect and Reject yielded the largest effect sizes. Difference was significant with respect to father’s Loss, with higher scores for trans women .The intensity of different traumatic experiences suffered by the GD and control subgroups, as yielded by the ComplexTQ scales, is shown in trans men and female controls, after the BH correction, yielding large effect sizes, trans men showed significantly higher scores on the following scales: fathers’ Neglect, Reject and Separation, and mothers’ Psychological Abuse. Moreover, higher scores for trans men were found with respect to the Loss of other figures .In the comparison between trans women and trans men, no significant differences were observed in the occurrence of trauma, the classification of polyvictimisation or the frequency of traumatic experiences, as examined through each ComplexTQ scale.With reference to the comparison within the GD sample between t], showing the poor emotional support and availability of fathers of GD persons. No significant difference was found in the comparisons between control males and trans women and between control females and trans men, nor between trans women and trans men.To explore loving parental caregiving, we considered the mean scores of the AAI Loving scale. A significant difference was observed between GD and controls with respect to the father’s relationship [GD = 3.11(1.59), Controls = 4.16(1.36); trans women and trans men and experiences of trauma between trans women and male controls and between trans men and female controls. In the current section, summaries of the main findings are presented for the reader’s convenience.In the current study, we explored the attachment representations and narratives and early relational history of a sample of GD adults at two levels of analysis. The first level considered the entire GD sample and compared this sample with controls and other samples from the literature; the second level narrowed the investigation to GD subgroups, comparing attachment and early relational experiences between Concerning the first level of inquiry (into attachment), it is noteworthy to underline that almost half of our sample (46%) was marked by a high percentage of U and CC classifications, indicating a prevalence of insecurity. In the control group, only one form of disorganization was found: the U profile with regard to loss, due in most cases to recent deaths. In contrast, the GD sample showed all three forms of disorganized profiles .In this regard, the results showed that our GD sample was significantly aligned with Italian  and inteSignificant differences were also found in specific AAI state of mind scales: our GD group showed higher Anger toward fathers and, to a lesser extent , Derogation toward fathers and Idealization toward mothers, relative to controls.trans children’s separation anxiety from their mother and their fantasized connection to her and With regard to these comparisons regarding state of mind scales, our aim was not to typify our individuals as belonging to specific attachment classifications, rather to compare representational ‘stances’ between groups and subgroups. The emerged polarized representations of parental figures are worthy of notice, since they add another aspect of unbalance to the participants’ attachment narratives regarding representations of their parents. As mentioned above, psychological and psychoanalytical theories have mostly seen family dysfunctions as etiological factors of transsexual conditions. In contrast, we took an agnostic approach to causation, considering insecure and disorganized attachment patterns – as well as adversity in early relationships – as potential concurrent risk factors for GD persons’ psychological development. Despite resounding psychoanalytical etiological accounts such as Coates’  focus ontrans participants (56%), we observed a co-occurrence of multi-type maltreatments during childhood, leading to polyvictimisation . Trans women, compared to control males: (1) were more neglected by both parents; (2) had more involving, rejecting and physically and psychologically abusive fathers; and (3) suffered more frequently from an early loss of the father. On the other hand, trans men, compared to female controls: (1) were more often victims of intensive rejection, neglect and early separation from fathers; (2) had more psychologically abusive mothers; and (3) prematurely experienced more losses of close relatives and friends.Regarding trauma history, whilst no difference emerged from the comparison within the GD group, interesting findings emerged from comparisons of the GD sample and controls, when divided according to natal gender . Second, findings should be generalized cautiously also by reason of the lack of a control group paired for age, numerosity and education. Finally, in this study, trauma history was assessed with the clinician-report ComplexTQ, as applied to AAI transcripts; therefore, trauma was not evaluated from the attachment measure through an independent source. However, as As mentioned above, the aim of the study was not to capture the causal relationships between trauma, attachment and GD. Rather, we considered both trauma and GD multi-dimensional constructs requiring no reductive or short-sighted attempts at explanation. Further research, however – likely using longitudinal designs – might explore more deeply the influence of early traumatic experiences and disorganized attachment representations on the development of a non-conforming gender identity.GG: Conception of the work, data collection, data analysis and interpretation, drafting the article. RV: Conception of the work, data collection, data analysis and interpretation. CM: Data analysis and interpretation, drafting the article. AF: Data collection, data analysis and interpretation, drafting the article. LC: Data collection. VL: Conception of the work, data analysis and interpretation, critical revision of the article. AS: Conception of the work, critical revision of the article, final approval of the version to be published.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.


Gossypium hirsutum (upland cotton) includes both domesticated varieties and wild populations of the same species. Most studies on mating systems describe cultivated cotton as self-pollinated, while studies on pollen dispersal report outcrossing; however, the mating system of upland cotton has not been described as mixed and little is known about its wild relatives. In this study we selected two wild metapopulations for comparison with domesticated plants and one metapopulation with evidence of recent gene flow between wild relatives and the crop to evaluate the mating system of cotton’s wild-to-domesticated complex. Using classic reproductive biology methods, our data demonstrate that upland cotton presents a mixed mating system throughout the complex. Given cotton’s capacity for outcrossing, differences caused by the domestication process in cultivated individuals can have consequences for its wild relatives. This characterization of the diversity of the wild relatives in their natural distribution, as well as their interactions with the crop, will be useful to design and implement adequate strategies for conservation and biosecurity.The domestication syndrome of many plants includes changes in their mating systems. The evolution of the latter is shaped by ecological and genetic factors that are particular to an area. Thus, the reproductive biology of wild relatives must be studied in their natural distribution to understand the mating system of a crop species as a whole.  Plant domestication is a complex and continuing process . For 10,Carica papaya), which is hermaphroditic, while native varieties and wild papayas are dioecious  or autogamy (self-fertilization).In Mesoamerica, ed forms ; hence, ed forms  – the coed forms  and geneed forms . Moreoveferences . Wegier G. hirsutum L., is a species with wild, feral, and semi-domesticated populations . Hand-pollination treatments .Sampling was performed in coastal dunes and dry forests of Mexico, in three of the eight wild cotton metapopulations defined genetically, geographically, and ecologically by eatments  were carTable 1), anticipating the risk of collecting too few fruits afterward: assisted self-pollination, automatic self-pollination, assisted cross-pollination (Table 1). The treatments that did not include bagging before anthesis were bagged after flower closure to help control for mechanical damage from bagging.In order to execute the hand-pollination treatments to test for different mating systems, a search was conducted for flower buds before anthesis . In eachlination , emascul®, Merck) solution and covered with a damp cotton swab; tissue culture lids were used. Seeds were checked daily until all reached emergence of the radicle. While some studies on seed germination consider only a set of seeds  was calculated for each study site following The outcrossing rate  and outcrossing (Wx) treatments, i.e., automatic self-pollination and emasculated control, respectively. For CPM, Wx was obtained with the fruit-set from the assisted cross-pollination treatment, because none of the emasculated control results were found when revisiting the metapopulation for fruit collection.where post hoc test was performed to evaluate the significance of the results. To compare germination frequencies and percentage of fruit-set, a chi square test was used with the post hoc standardized residue test for each one. Outliers were identified using the method described by lme4, multcomp, stats, and ggplot2 packages of R version 3.4.3 . The CPM open-pollinated control showed the highest value of all treatments among all groups; YPM showed the highest fruit-set produced by outcrossing, and the lowest by automatic self-pollination and control treatment. On the other hand, the highest fruit-set was observed for all the treatments in SPM, with exception of the open-pollination control.All treatments produced fruits regardless of the metapopulation . The average number of seeds per fruit was 15.9 in SPM, 15.4 in domesticated, 12.3 in CPM, and 10.0 in YPM, while the average number of ovules was 16.3 in SPM, 28.6 in domesticated, 15.6 in CPM, and 13.4 in YPM. Regarding seed-set, the control treatment of domesticated cotton was lower than that of wild and introgressed plants  . When evaluating the results of each metapopulation individually, CPM presented seed-set differences between the control and the rest of the treatments (P(χ2) = 0.1 × 10-4, df = 3), in SPM the differences were found between cross-pollination and all treatments, except emasculated control , while in YPM and the domesticated there were no significant differences among treatments . On the other hand, seed weight only presented differences in SPM, between cross-pollination and both assisted and automatic self-pollination  . In YPM, CPM, and the domesticated cotton, no significant differences among treatments were observed .Seeds were produced both by outcrossing and selfing treatments in all metapopulations (Table 3).Less than 20% of the seeds from wild metapopulations CPM and YPM germinated. Regarding the domesticated group, 40–63% of the seeds germinated, except for the seeds produced by cross-pollination that only reached 28%. The seeds from the five treatments assessed at SPM showed germination percentages above 83%, with cross-pollination reaching the highest value of 96% , the slope of the curve suggests that wild upland cotton presents some kind of inhibition to the completion of germination, whereas domesticated populations do not display this behavior. As shown in Figure 4A, domesticated seeds germinated faster, within the first 6 days, whereas seeds presenting evidence of introgressive hybridization (SPM) reached 95% of germination within the first 7 days and continued germinating for 48 days. Unlike domesticated and SPM seeds, the seeds of wild plants germinated over the course of 73 days . Concerning the pollination treatments from all study sites, 50% of the seeds of all treatments germinated within the first 5 days; however, after the 5th day the difference in germination rate is evident between autogamy and the rest of the treatments .With regard to the germination rate of the seeds that germinated (Te > 0), which is indicative of a mixed mating system. Wild metapopulations (YPM and CPM) recorded a higher outcrossing rate  than SPM (0.40) and domesticated (0.65) ; thus, the analyzed plants have the capacity to produce progeny by both autogamy and xenogamy. To discard geitonogamy, it is necessary to perform a molecular genetic analysis of paternity. However, since autogamy is common in our system, there is no need to discard this type of allogamy. Furthermore, previous studies , which indicates that when the value is 0.5 the mating system is equally balanced between self and cross-pollination. Any value different from 0 (completely self-pollinated) or 1 (completely cross-pollinated) implies a mixed mating system; when Te > 0.5, the system is predominantly allogamous-xenogamous, whereas when Te < 0.5, the system is predominantly autogamous. Our observed rates vary from Te > 0.5, e.g., 0.71 (CPM), 0.72 (YPM) and 0.65 (domesticated), to Te < 0.5, e.g., 0.40 (SPM). Domesticated plants, and wild CPM and YPM, have a greater contribution of seeds from cross-pollination in the next generation, although the contribution of self-pollination is high and important, and it contributed to maintenance of genetic structure. The high contribution of self-pollinated seeds in SPM is striking, far from being similar or intermediate between wild and domesticated; local factors may be affecting the result and should be addressed in a future study.Figure 4B). As suggested theoretically, when germination does not differ among treatments, self-pollination is not the cause of inbreeding depression  among autogamy and the other treatments is observed. Such discrepancy is due to the difference in number of seeds produced in each treatment  and high FST. Self-pollination and cross-pollination seem to maintain the genetic diversity of the species in the wild, although crossings with domesticated members of the complex .Finally, one of the fitness components measured in plants is seed weight , due to Our analyses show differences in characters linked to some of the reproductive structures of upland cotton, which can be associated with the domestication syndrome and will be discussed below.P(χ2) = 0.001, df = 2], which was initially estimated to obtain the seed-set in each population  Walp.] into two different groups: cultivars able to flower early under inductive conditions, with ovule number lower than 17  and cultivars not able to do so, with ovule number higher than 17 . Moreover, qon1 and qon3) that regulate ovule number in cowpea; the alleles from the wild parent increase this trait as opposed to the cultivated, which reveals a feature of cowpea’s domestication syndrome. In the case of climbing common bean (Phaseolus vulgaris L.), among the changes that occurred during the domestication process is the modification on the number of ovules, which changed from 5–8 to 2–9 ovules  produce, proportionally, a lower quantity of seeds, which implies that they are not efficiently using the resources invested on ovule production .One of the traits selected for during domestication is rapid germination , as thisFigures 3); its fruits produce more seeds than the other populations  . These characteristics can have demographic consequences in the short term, unless there are other factors that regulate this growth. On the other hand, contrary to what was expected for SPM, their resemblance to domesticated seed germination is higher than with the wild ones. The behavior is also dissimilar for introgressed seeds, which took longer to complete germination than domesticated and wild seeds . This last phase displays a very slow response in SPM, probably associated with the loss of physiological responses, resembling domesticated plants. It is important that these analyses are repeated in subsequent years, to confirm if there is an eco-genetic trend . These hybridization events could lead to a decline in wild genetic diversity, as opposed to native semi-domesticated varieties in traditional Mesoamerican systems where there is evidence that domesticated genomes have formed not only by selection under domestication, but also by gene flow with other closely related populations and species (Gene flow between crops and wild relatives should be examined on a case-by-case basis , especia species . For thi species .G. hirsutum, along with factors modified by traditional genetic improvement and modern genetic engineering, will be problematic for agroecosystems (In this study we found that the reproductive capacity of introgressed cotton is greater than that of wild and domesticated plants. This reveals a scenario that osystems  and ecososystems . Cotton osystems , so it wG. hirsutum within its natural distribution. Some of the factors that have an effect on the results can be associated with the environment (pollen viability, nectar production, and pollinator activity due to environmental conditions; G. hirsutum and, consequently, conservation strategies should take this key interaction into consideration.Finally, local conditions can influence the results of reproductive biology studies ; hence, G. hirsutum in its natural distribution, but it is in agreement with previous studies in populations of domesticated cotton (Supplementary Material G. hirsutum should be considered as having a mixed reproductive strategy throughout its whole complex, rather than being primarily autogamous. Management strategies and policies meant to conserve the diversity of cotton’s wild-to-domesticated complex must take this into account.This study found that upland cotton’s wild-to-domesticated complex presents a mixed mating system. This information is new for wild, domesticated, and introgressed Furthermore, physiological differences were found between cultivated cotton and its wild relatives, especially in traits such as the number of ovules per flower, number of viable seeds per fruit, and their germination behavior. Given the evidence of gene flow and introgression, these traits should be monitored systematically in wild populations and agroecosystems of interest for conservation, as well as the impact on ecological interactions, such as pollination. On the other hand, the diversity contained in the wild-to-domesticated complex must be included in long-term conservation strategies, so that future generations can have access to genetic resources with greater chances of surviving the changing environments.AW, VA, and RV-L designed the research, participated in fieldwork, performed the analyses, and wrote the manuscript. AW coordinated the study. WK designed the fieldwork and revised the analyses. AP-M participated in fieldwork and performed the analyses. JP-L, VV-B, and DA-L conducted the analyses. All authors analyzed the results and wrote the 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.