Reproductive efficiency stands as a critical determinant of profitability within beef production systems. The incorporation of molecular markers can expedite advancements in reproductive performance. While the use of SNPs in association analysis is prevalent, approaches centered on haplotypes can offer a more comprehensive insight. The study used registered Simmental and Simbrah cattle genotyped with the GGP Bovine 150 k panel. Phenotypes included scrotal circumference (SC), heifer fertility (HF), stayability (STAY), and frame score (FS). After quality control, 105,129 autosomal SNPs from 967 animals were used. Haplotype blocks were defined based on linkage disequilibrium. Comparison between haplotypes and SNPs for reproductive traits and FS was conducted using Bayesian and frequentist models. 23, 13, 7, and 2 SNPs exhibited associations with FS, SC, HF, and STAY, respectively. In addition, seven, eight, seven, and one haplotypes displayed associations with FS, SC, HF, and STAY, respectively. Within these delineated genomic segments, potential candidate genes were associated. Reproductive efficiency is essential for the profitability of beef production systems. High reproductive rates significantly increases the biological and economic efficiency of cow–calf production systems . The maiGenetic evaluations can expedite improvements in reproductive performance and other traits with low heritability . AdditioWhile SNPs are commonly used in association analyses, haplotype-based methods offer more comprehensive information. In contrast to SNPs, which can have a maximum of two alleles, haplotype blocks can encompass more than two haplotypes. Haplotype blocks consist of two or more closely located loci, making them prone to co-inheritance due to their high linkage disequilibrium. Haplotype-based methodologies, aimed at enhancing the efficacy of identifying causal haplotypes, are rational, considering that genes operate as coherent gene sets, rather than as individual SNPs within the set. These haplotype-based techniques are anticipated to provide improved control over false positives when compared to single-SNP methods as they concentrate on the complete haplotype block rather than on the individual SNPs within that block . HaplotyThe ability to replicate the regions is associated with the economically significant traits reported in other studies ,9,10,11,Also, a multi-breed reference population has the potential to significantly enhance the precision of genomic predictions by enabling breeds with limited reference population data to leverage information from other breeds . NotablyThe objective of this study was to compare the results of an association analysis with haplotypes and SNPs for reproductive traits and frame score (FS) in Simmental and Simbrah cattle using the EMMAX, BayesB, and BayesC methods.Frame score (FS): Hip height converted to frame score represents a linear measurement employed by cattle producers to assess the potential lean-to-fat ratio of an individual animal within a performance-oriented program. Calculated using the reference tables provided by the Beef Improvement Federation .Scrotal circumference (SC): For yearling bulls, scrotal circumference measurement entails encircling the broadest section of the scrotum with a scrotal tape while the testicles are in a fully extended state.Heifer fertility (HF): Represents the probability that the daughters of a sire have their first calving at the age of 3 years or earlier.Stayability (STAY): Indicates the probability that the daughters of a sire, having had a calf before 3 years of age, will have at least a second calf before 6 years of age.The phenotypes employed in the current study were:Estimated breeding values (EBV) for all traits were computed using data from the Mexican Simmental-Simbrah Breeders Association, which was subsequently submitted to Mexico’s National Institute for Forestry, Agriculture, and Livestock Research (INIFAP) for the National Genetic Evaluation.In line with the protocols detailed by Garrick et al. , these EBlood samples were obtained from each of the animals participating in the study. These samples were individually labeled and subsequently dispatched to Neogen’s GeneSeek Laboratory, located in Lincoln, NE, USA, for the purpose of DNA extraction and genotyping. The GGP Bovine 150 k chip, consisting of 138,962 SNP markers, was employed for genotyping. All SNPs with a call rate lower than 0.95 and a minor allele frequency below 0.05 were excluded. Furthermore, individuals with a call rate lower than 0.95 were removed from the dataset. Following the quality control assessment, a total of 105,129 autosomal SNPs from 967 animals  were employed for genotype association tests .Haplotype blocks were defined as regions smaller than 200 kb, with at least 90% of the comparisons between SNPs within the block in “strong LD”. The 90% confidence interval for LD was established between 0.70 and 0.98. A total of 16,651 haplotype blocks (each block had between 2 and 48 SNPs) containing 57,202 haplotypes were inferred and used for the association analysis. Quality control and haplotypes were determined with Plink 1.07 .A comparison was made between haplotypes and SNPs for reproductive traits and FS in Simmental and Simbrah cattle. The association analyses were carried out separately for each breed, combining the information of both breeds . Two different approaches were used, a single-marker GWAS and two Bayesian GWAS.For the single-marker GWAS, EMMAX was implemented using SNP & Variation Suite 7 ; this is a single-locus mixed model GWAS approach that fits a genomic relationship matrix to account for genetic covariance among animals . In singp-values for the Bonferroni-corrected thresholds at the suggestive and 5% genome-wide significance levels were determined by dividing 1 and 0.05, respectively, by the total number of markers utilized in the GWAS. The concept of the suggestive level was originally introduced by Lander and Kruglyak [The Kruglyak  and signKruglyak . FollowiThe Bayesian models were implemented via the BGLR statistical package of the R program . Two a pFor a continuous response  serving as the pivotal parameter for this adaptation. This adjustment relies on a Student-t approximation for posterior inference derived from the simulations ,R^c=d+3Significant disparities in these variances signal non-convergence (2 = 0.2) previously observed in beef cattle [Bos taurus ARS-UCD1.2 genome assembly [The SNP and haplotypes associated were mapped at 100 kb intervals on both sides, given the average LD  plots did not reveal any substantial deviations . The cloIn the joint association analysis of FS with SNPs, 15 associated regions were located on chromosomes 4, 5, 6, 7, 8, 11, 16, 17, and 22 . Only onIn the Simmental data analysis with SNPs, six associated regions were found on chromosomes 2, 17, 19, and 20 . The assFor Simbrah cattle, associated regions were found on chromosomes 3, 17, and 18 . There wIn the haplotype association analysis with the joint analysis, two regions were identified on chromosomes 4 and 8, both with the BayesC model . In addiIn the haplotype analysis of the Simbrah cattle, only one region on chromosome 3 was identified with the three models . In SimmBy contrasting the regions associated with SNPs and haplotypes, we found two regions in common in the joint analysis . For theAccording to the joint association analysis of scrotal circumference with SNPs, seven associated regions were located on chromosomes 1, 3, 6, 10, and 17 . With thTwo associated regions were found on chromosomes 10 and 13 in the association analysis with SNPs for Simmental cattle . All modThere were associated regions on chromosomes 6, 10, 17, and 23 for Simbrah cattle . On chroOn chromosomes 19 and 25, the joint analysis revealed two haplotype associated regions . Each ofThe haplotype analysis of Simbrah cattle identified two regions on chromosomes 2 and 23 . With alFour regions were found on chromosomes 2, 8, 9, and 13 of Simmental cattle . ChromosBy contrasting the regions associated with SNPs and haplotypes, we found no regions in common in the joint analysis. For the individual analyses of Simmental and Simbrah, one region was found to be common in each case. In addition, there was a region in the SNP analysis in common between Simmental and Simbrah with the joint analysis in chromosomes 10 and 6, respectively.The joint association analysis of heifer fertility with SNPs identified four associated regions on chromosomes 4, 17, and 18 . With thIn the SNP association analysis for Simmental cattle, two associated regions were found on chromosomes 4 and 21 . The assBased on the haplotype association analysis with the joint analysis, four regions were identified on chromosomes 2, 4, 15, and 17 . Two regThe haplotype analysis of Simbrah cattle identified only three regions on chromosomes 2, 8, and 28 . All regComparing the regions associated with SNPs and haplotypes, we found one region in common. Additionally, the Simbrah and joint analyses shared a region on chromosome 17.In the joint association analysis of STAY with SNPs, one associated region was located on chromosome 21 with the BayesC model . In SimmOur analysis of Simmental individual analyses of chromosome 28 revealed a region common to both haplotypes and SNPs .In the Simmental data analysis, within the SNP windows on chromosome 2, QTLs were previously associated with a body form composite index in Holstein cattle; this index contemplates the height of the animal, which is a measure that is also used to calculate FS . Also, iFrom the results of the joint model, several coincidences were found within the regions associated with FS, with the databases of QTLs. On chromosome 5 and 7, associations with SC in Angus cattle  were fouThe ligand-dependent nuclear receptor corepressor-like (LCORL) gene on chromosome 6 has been reported to be associated with body frame size (height) at puberty in Japanese Black and Charolais x German Holstein cattle ; it is tIn the Simbrah data analyses, two QTL associated with body weight (yearling) inside of the haplotypes windows were found on chromosome 2 in Angus × Brahman cattle  and 23 iIn Simmental cattle, associations with body weight (yearling) were found within two haplotype windows on chromosomes 8 in crossbreed cattle  and 13 iIn the joint-data analysis, associations were found in two SNPs windows on chromosome 1 in Angus × Brahman cattle  and 17 iIn Iranian Holstein cattle , on chroIn the Simmental data analysis, within the SNP windows on chromosome 21, QTLs were previously reported to SC in Angus cattle , which iβ 4 (CACNB4) gene associated with calving to first service was identified, which is the same gene that was associated in the present work with HF  on chromosome 4 had been reported to the retained placenta signals with associations related to milk production, productive life, and health and reproduction traits, including calving ease and stillbirth ,39. In HIn goats, the expression of the CKLF-like MARVEL transmembrane domain-containing 2 (CMTM2) gene on chromosome 18 was more elevated in the ovaries of multiple prolific goats at first kidding compared to non-prolific goats .In the Simmental data analysis, within the SNP windows on chromosome 1, QTLs were previously reported for the interval to first estrus after calving in Holstein cattle . With thIn Costeño con Cuernos cattle from Colombia, on chromosome 21, the cathepsin H (CTSH) gene associated with age at first calving was identified , which iAs shown in the results, none of the SNPs crossed the threshold level at the genome level for the EMMAX model. Nonetheless, this is not an isolated case, as in many studies, single locus GWAS models, such as EMMAX, did not significantly exceed the threshold of significance, so they resort to other methodologies . AlthougAccording to our dataset, while using a single marker with EMMAX makes it more stringent to assert significance, employing Bayesian models offers a superior understanding of how LD affects the outcome. However, as Legarra et al.  mentionsp) is significantly larger than the number of samples (N). Consequently, conventional methods of maximizing the likelihood based on effect sizes often result in severe overfitting. To address this problem, a common approach is to introduce a regularization term into the log likelihood, encouraging many components of β (effect sizes) to approach zero. From a Bayesian perspective, this can be viewed as maximizing the posterior distribution p(β|X) [In GWAS, a frequent issue arises when the number of parameters (n p(β|X) . This adIn the present study, we identified several significant SNPs and favorable haplotypes using SNP and haplotype-GWAS approaches, respectively, that regulate FS, SC, HF, and STAY in Simmental and Simbrah cattle. The idea of utilizing haplotype-based GWAS has been suggested as an additional method to enhance the advantages gained from LD . This isSNP- and Hap-window approaches to identify important genomic regions accounted for LD and better characterized QTL than the individual effects . For allAssociation analyses have identified many common genetic variants associated with traits of interest, but these variants typically have small effect sizes and account for only a small fraction of the heritability of most traits. This missing heritability revealed in many well-powered analyses associations suggests that there are many important architectural details of traits that remain to be discovered. However, an association that explains only a small proportion of heritability may nonetheless provide valuable biological insights ; therefoOur study provides new insights into the genetic basis of reproductive traits and FS in Simmental and Simbrah cattle. We identified positional candidate genes with functional evidence that could improve bull genomic prediction for these traits. It has been proven that GWAS-selected sequence variants improve genomic prediction reliability , which cA total of 23, 13, 7, and 2 SNPs were associated with FS, SC, HF, and STAY, respectively. Also, seven, eight, seven, and one haplotypes were associated with FS, SC, HF, and STAY, respectively. Through these regions, we identified possible candidate genes for the different traits used in this study. These regions may be useful in Mexican Simmental and Simbrah cattle. No regions were found to be common between Simmental and Simbrah cattle, which indicate that we are dealing with two completely different genetic architectures.


Transmission electron microscopy was utilized for morphological characterization. BTO nanoparticles obtained have spherical shapes with orthorhombic structure and the average size distribution depended on the liquid media nature. In alcohols, BTO NPs were spherical with a carbon layer around them. X-ray diffraction, UV-Vis absorption spectra, and X-ray photoelectron spectroscopy were used to confirm the structural, optical, and elemental properties of the ablated products. The presented results show that PLAL is a viable technique for the synthesis of high-quality BTO nanoparticles with enhanced optical properties for possible applications in photocatalysis.Bismuth titanate (BTO) nanoparticles were obtained by pulsed laser ablation in liquid media (PLAL). Distilled water, ethanol, isopropanol, and acetone were used as media for laser ablation experiments, in which the colloidal solutions were obtained. Laser ablation was carried out using the second harmonic and fundamental wavelength of a pulsed Nd:YAG laser  with laser fluences of 25 and 12 mJ/cm Be layers ,17,18 ane layers , coprecie layers , hydrothe layers , sonochee layers , and mece layers ,24, amonOn the other hand, pulsed laser ablation in liquid (PLAL) is a novel method for the generation of particles with sizes ranging from the micro- to nanometer scale directly from bulk materials. This technique consists of a pulsed laser focused on a bulk material, which is immersed in a liquid medium. The mechanism of nanoparticle generation by PLAL starts with the interaction of the bulk material with a laser pulse that has a fluence greater than its ablation threshold ,26, whicOn the other hand, laser parameters, as well as the physicochemical properties of the target material and liquid medium, play an important role in the stages of nanoparticle generation ,38; for In this work, BTO nanoparticles were synthesized using pulsed laser ablation in different liquid media (distilled water (DW), ethanol, isopropanol, and acetone) and their morphological, structural, elemental, and optical properties were studied.2 for the laser operating at the fundamental wavelength and its second harmonic, respectively. A system of linear translation was employed to avoid local ablation and increase NP production and efficiency; the translation speed was set to 500 µm/s. The time of ablation was 1 min for every experiment. BTO nanoparticles (NPs) were obtained using the pulsed laser ablation in liquid media (PLAL) technique. For the synthesis process, a Nd:YAG LQ 929 pulsed laser, , was used at its fundamental wavelength (1064 nm) and its first harmonic (532 nm), with a frequency of 10 Hz and 10 ns pulse width . A target of bismuth titanium oxide (BTO) was used to produce BTO NPs in distilled water, ethanol, isopropanol, and acetone to evaluate the properties of the ablated products. The BTO target was immersed in 50 mL of each liquid in a glass beaker of 200 mL. The laser beam was focused perpendicularly to the target surface using a mirror (99% reflective) and a concave lens of 50 cm focal length. Laser fluence was calculated to be 25 and 12 mJ/cmIn our work, the dark or tenuous nature of the solution is indicative of the concentration of the nanoparticles but not the size. Likewise, a brown color was observed in liquid media such as alcohols and in organic media; this could correspond to compounds that were formed (between organic media and NPs).Morphology and crystal structure were analyzed using a TEM microscope, model FEI Titan G2 80-300 . To obtain TEM micrographs, a drop of each colloidal solution was placed in a TEM grid after ablation. To study crystal structure and elemental composition, precipitates of the obtained solutions were taken and placed in a silicon wafer and dried at room temperature until sufficient material for both analyses was gathered. X-ray diffractograms were obtained in a PANAlytical Empyrean diffractometer  using Cu K4Ti3O12 (JCPDF No. 73-2181), while the nanoparticles produced in ethanol showed no planes, due to the amorphous nature of nanoparticles obtained by this medium. Moreover, BTO nanoparticles synthesized by PLAL using For the nanoparticles produced using There is an excellent review on the variables that determine particle size, morphology, and properties of the resulting colloids via laser ablation in liquid media . In shor= 532 nm a for DW,Those produced in acetone and isopropanol have a preferred orientation in the (1 1 7) direction; see 4Ti3O12 target are: 158.78 eV for Bi 4f7/2, 458.2 for Ti 2p3/2, and 529.87 eV for O 1s, which are values in agreement with similar materials. It is important to note that a component for the Bi 4d5/2 core level appears on the Ti 2p spectra, which is expected for BTO, with a binding energy of 466.2 eV [7/2 and O 1s, respectively. This is due to the solvent effects and laser energy. Some of the peaks of Bi 4f also possess a metallic component of 157.95 eV. These effects can be explained by a strong solvent effect and the high photon energy of the laser. Usually, properties such as refraction index, thermal properties, and flash point influence the final desired result of the nanoparticles [466.2 eV . The pea466.2 eV . After a466.2 eV . It is iarticles . It is iid media . As suchid media . Some Biid media .g) of the BTO NPs was studied (inset inside of 3CH3), since the energy radiation interacts with the bonds of acetone, absorbing energy at these wavelengths as reported in [This signal is characteristic of acetone  confers high reactivity to the medium in this wavelength range donating electrons that react more with metals.UV-Vis spectroscopy was employed to study the optical absorption of the ablated BTO nanoparticles at both = 532 nm a and λ = 1064 nm b. FigureThese results show that the BTO NPs produced by PLAL could have potential applications in photocatalysis, since their optical transition energy could be provided by the solar spectrum.Bismuth titanate nanoparticles have been successfully synthesized via pulsed laser ablation in distilled water, ethanol, isopropanol, and acetone. The morphological, crystalline, and optical properties have been studied to evaluate the impact of the laser wavelength (532 nm and 1064 nm) and liquid medium on the properties of the BTO nanoparticles. TEM micrographs show that PLAL produced spherical particles, yet the sizes of the nanoparticles depended on the medium. SAED and XRD confirmed the polycrystalline nature of the BTO nanoparticles, with peaks associated with the orthorhombic phase of bismuth titanate. XPS spectra verified the chemical state of Bi, Ti, and O of each sample. Optical absorption spectra showed differences between the BTO NPs produced in different media and at different laser wavelengths. The optical bandgap values lie between 2.27 and 3.27 eV, depending on the medium and laser wavelength used.


PANK2) gene, is the most common neurodegeneration with a brain iron accumulation (NBIA) group. The “eye of the tiger” sign in the magnetic resonance imaging demonstrated a bilateral hyperintense signal in the basal ganglia region on T2-weighted images, which is a characteristic feature of the diagnosis. PKAN is classified into 2 main types. The early-onset type (classic type) with rapid progression is characterized by symptoms of gait impairment and dystonia leading to loss of ambulation in early childhood. In the later-onset type , slow progression usually takes place in the second decade of life with symptoms of neurodegeneration, dystonia, dysarthria, rigidity, choreoathetosis, and motor impairment. Until now, PKAN patients have only been reported in a few countries in Asia such as China, Korea, India, Iran, Taiwan, and Thailand.Pantothenate kinase-associated neurodegeneration (PKAN), also called Hallervorden-Spatz syndrome, is a rare autosomal recessive disease associated with brain iron accumulation and characterized by progressive dystonia, dementia, and dysarthria symptoms. PKAN, caused by a defective pantothenate kinase 2   that has been confirmed as the cause of the disease.Whole exome sequencing was performed to identify heterozygous mutations in the In this study, the first Vietnamese patient with late-onset PKAN was diagnosed by the whole exome sequencing method.The patient’s case marks an important milestone for the first case in Vietnam. The results of the study will provide a scientific basis for clinicians in the diagnosis and genetic counseling of patients. PANK2), PLA2G6, and FA2H. PKAN  is a rare genetic disorder characterized by iron deposition in the basal ganglia region and has early onset before age 10.Pantothenate kinase-associated neurodegeneration  is the most common type of neurodegeneration with brain iron accumulation (NBIA) group. NBIA is a group of heterogeneous neurodegenerative disorders including symptoms such as postural disturbances , abnormal motor, gait impairment, visual impairment, dystonia, dysarthria, and neurodegeneration. Neurodegeneration is detected in specific regions of the central nervous system leading to abnormal progressive involuntary motor symptoms, loss of walking ability, mental confusion, intellectual disability, joint stiffness, and retinitis pigmentosa. NBIA is characterized by radiographic evidence of iron accumulation in the brain and typically begins in childhood. NBIA is classified into separate disorders based on genetic causes and related genes such as the pantothenate kinase 2  and increased deep tendon reflexes (in one-quarter of patients), whereas a few (about 8%) had acanthocytes and aberrant plasma lipoproteins. The classic form has a mean age of onset of 3 to 4 years with symptoms of postural disturbances, progressive extrapyramidal, and corticospinal tract symptoms. Most of these patients develop motor impairment between the age of 5 and 8 and loss of ambulation within 10 to 15 years. Extrapyramidal symptoms, dystonia, dysarthria, stiffness, neurological manifestations, attention deficit hyperactivity disorder, chorea, Parkinson, and cortical symptoms have also been reported in these patients.The incidence of PKAN is estimated at 1 to 3: 1000,000. Neurobehavioral abnormalities, psychosis, intellectual disability, cortical symptoms, gait impairment, and extrapyramidal symptoms were also observed in these patients. In these patients, dystonia was the most common symptom (87%), in which the muscles of the upper and lower limb muscles extremities were affected leading to axial dystonia, difficulty walking, tremors, and chorea. Other symptoms such as spasticity, increased deep tendon reflexes, and plantar extensor reactions occurred in 25% of patients and cognitive impairment was detected in 29% of patients. Visual impairment and retinal degeneration could be present with less frequency in these patients. The atypical form shows a slower progression than the classical form. The ability to walk could be lost in time from 15 to 40 years after appearing the first symptom.Later-onset atypical form (10 years of age) with symptoms of speech and mental disorders, dystonia, spasticity, increased deep tendon reflexes, and chorea (accounting for 25% of patients). The clinical feature of neurodegeneration due to iron accumulation in the globus pallidus resembling the “eye of the tiger” sign on the magnetic resonance imaging (MRI) of the affected individuals is considered a clinical feature specific to the disease. MRI brain demonstrated iron accumulation in the brain through a low signal within a hyperintense region in the globus pallidus area on T2-weighted image in both the atypical and classic form.,5 Other types of neurodegeneration due to brain iron accumulation that are not related to the PANK2 gene mutations usually do not exhibit the “eye of the tiger” sign. Thus, a diagnosis is made based on the clinical history and MRI imaging. Recently, with the advantages of next-generation sequencing, the application of whole exome sequencing (WES) has been widely used to improve efficiency in diagnosis.Until now, there is no specific treatment for PKAN. Treatment requires a combination of specialties including neurologist, ophthalmologist, physiotherapist, and speech therapist.PANK2 gene , encoding the enzyme that catalyzed the phosphorylation of pantothenate (vitamin B5) in the bioavailability of coenzyme A (CoA), was identified as the causative gene of the PKAN disease. The PANK2 gene, present in the mitochondria and the nucleus, is located on chromosome 20p13 and spans approximately 34 kb with 7 exons.,8–10 The PANK2 gene encodes an important regulatory enzyme (consisting of 571-amino acids) among four pantothenate kinase enzymes that are targeted to mitochondria of CoA biosynthesis. The formation of CoA is essential for energy metabolism, fatty acid synthesis, degradation, and synthesis of many neurotransmitters as well as glutathione, an antioxidant that prevents cell damage caused by reactive oxygen species. The deficiency of CoA in energy production can lead to forming of reactive oxygen species free radicals and damaging cells. Inactivation of the PANK2 protein leads to the accumulation of cysteine-containing substrates and iron-induced lipid peroxidation which is responsible for PKAN. In addition, cysteine is a substrate of the PANK2 enzyme and may play a key role in iron accumulation, therefore in PKAN patients, there is an increase of cysteine and chelate leading to the accumulation of iron in the brain.,12 It is thought that excessive accumulation of cysteine in the globus pallidus region results in decreasing pantothenate, causing iron chelation and oxidative damage to basal ganglia. However, how a defect in mitochondrial CoA synthesis leads to iron accumulation and the specific PKAN phenotype remains unclear.The ,13–24 In this article, we report the first PKAN patient in Vietnam due to the PANK2 mutations.NBIA caused by PANK2 mutations has been reported previously in Asian ethnic groups, for example, Chinese, Korean, Indian, Iranian, Taiwan, and Thailand.The patient is a 12-year-old boy who presented with a three-year history of muscle weakness, gait disturbance, dysarthria, and dysphagia since the age of 10. Other clinical symptoms observed include difficulty walking, easy falling, skinny body, reduced vision, difficulty holding a pen, and difficulty chewing and swallowing. Evidence of iron accumulation in the brain as the “eye of the tiger” sign detected on the MRI showing a hyperintensity in the globus pallidus region on T2-weighted images . The consent form was written by the patient’s parents for the publication of any potentially identifiable images or data included in the article. The post-alignment process of data includes creating indexes, marking, removed repeated reads on the alignment bam file used by the Picard tool (https://broadinstitute.github.io/picard/command-line-overview.html). The GATK package version 4.1 was used for calling variants. Sanger sequencing was done for validating these 2 mutations in the patient and the members of his family. PCR amplification was carried out on an Eppendorf Mastercycler EP gradient . PCR products were sequenced on ABI PRISM 3500 Genetic Analyzer machine (Thermo Fisher Scientific Inc.). The sequencing data were analyzed using BioEdit 7.2.5 software. Two mutations  have been detected in the PANK2 gene (NM_153638.4) in a compound heterogeneous status. According to ACMG guidance , both mutations are reported as pathogenic. These mutations  have been registered in the SNP database (https://www.ncbi.nlm.nih.gov/snp/) with accession number rs137852962, rs1250997630 and ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) with accession number VCV000004551.3, VCV000667397.6, respectively. The heterozygous mutation c.856C > T, p.Arg286Cys was found in the father of the patient, and the heterozygous mutation c.1351C > T, p.Arg451Ter was found in the mother and old brother of the patient  following manufacturer guidelines. The DNA was used for WES on the Illumina platform (Illumina Inc.). The GRCh38 human genome was used as reference data for mapping the paired-end reads by BWA0.7.17 software. included all neurological disorders with progressive extrapyramidal symptoms and intellectual disability. The pathogenesis of the NBIA disorders as well as the role of iron in the causal of neurodegeneration has not been elucidated yet. Diagnosis of PKAN is based on clinical characteristics and anatomopathological findings. The classic form accounts for 75% of all PKAN cases with a mean age of onset at 6 years (range from 6 months to 12 years), the remainder is the atypical form with a mean age of onset of 14 years (range from 1 to 28 years). In this study, symptoms of gait disturbance, dystonia, dysarthria, dysphagia, reduced vision, and difficulty holding, chewing, and swallowing were observed in the first typical case of PKAN in Vietnam. However, several other features such as choreoathetosis, spasticity, Parkinson, tremor, and increased deep tendon reflexes that were reported in Asian patients but not in the Vietnamese patient. These features were observed more frequently in Asian patients than in Caucasians. Asian patients also have less complex presentations and a lower prevalence of pyramidal signs, mental impairment, and parkinsonism than Caucasians.Pantothenate-associated neurodegeneration (PKAN) is a form of the inherited progressive neurological disease called NBIA disorder, a term introduced by Hayflick et al in 2003, The PANK2 gene  was identified as the causative gene of the PKAN disease. PANK2 is a homodimer with each monomer including 2 conserved domains  and the long N-terminal region (with 200 amino acids). Dimerization is through long α-helices (aa 486–513) and 2 reaction regions on an extended loop (aa 446–471) and another helix (aa 414–426). The ATP binding site is located between domain A (aa 219–221) and domain B (aa 520–522) with active site E338 located in domain A.PANK2 mutations including insertion, deletion, and frameshift mutations in the PANK2 gene are linked to early-onset disease. In contrast, the missense mutations had in both early- and late-onset disease. The early-onset patients had missense mutations in the mitochondrial trafficking domain  of PANK2 and showed a decreased mitochondrial membrane potential with severity phenotype more than that from late-onset patients with mutations in the other 2 domains.Diagnosis is made based on the clinical history and MRI results (the “eye of the tiger” sign demonstrated iron accumulation in the brain), and recently, WES has been used to reduce the time and improve diagnosis efficiency.,21,28 The mutation of c.856C > T (p.Arg286Cys) in exon 2 has been previously reported in German patients with hand and gait dystonia and in Chinese patients who presented the symptoms of inarticulation, bradykinesia, gait instability, hyperextension. These mutations locate in the helices of the catalytic core region in the PANK2 protein. The mutation c.856C > T (p.Arg286Cys) locates at the β-strand domain (aa 278–288) and the mutation c.1351C > T (p.Arg451Ter) locates at the ATPase nucleotide binding site domain (aa 442–483). These sites are important for the activation of the PANK2 protein  mutation (exon 4) has been reported in Taiwan and Korean patients.PANK2 gene in the first Vietnamese patient with PKAN. The patient’s case marked an important milestone for the first case of the disease in Vietnam. The results provide evidence for a definitive diagnosis to orientate the treatments and genetic counseling for the patient.In this study, we performed WES analysis and identified a compound heterozygous mutation  in the We would like to thank the financial support by Vietnam Academy of Science and Technology for Institute of Genome Research, KHCBSS.01/22-24. We would like to express our deep gratitude to the patient who participated in this study.Conceptualization: Van Khanh Tran.Data curation: Chi Dung Vu, Hai Anh Tran.Formal analysis: Nguyen Thi Kim Lien.Investigation: Chi Dung Vu, Hai Anh Tran.Methodology: Chi Dung Vu, Hai Anh Tran, Nguyen Van Tung, Nguyen Ngoc Lan.Software: Nguyen Van Tung, Nguyen Ngoc Lan.Supervision: Nguyen Huy Hoang.Visualization: Van Khanh Tran.Writing – original draft: Van Khanh Tran, Nguyen Thi Kim Lien.Writing – review & editing: Van Khanh Tran, Nguyen Thi Kim Lien, Huy Thinh Tran, Nguyen Huy Hoang.


Several measurements have been used to predict the success of weaning from mechanical ventilation; however, their efficacy varies in different studies. In recent years, diaphragmatic ultrasound has been used for this purpose. We conducted a systematic review and meta-analysis to evaluate the effectiveness of diaphragmatic ultrasound in predicting the success of weaning from mechanical ventilation.Two investigators independently searched PUBMED, TRIP, EMBASE, COCHRANE, SCIENCE DIRECT, and LILACS for articles published between January 2016 and July 2022. The methodological quality of the studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool; additionally, the certainty of the evidence is evaluated using the GRADE  methodology. Sensitivity and specificity analysis was performed for diaphragmatic excursion and diaphragmatic thickening fraction; positive and negative likelihood ratios and diagnostic odds ratios (DOR) with their confidence intervals (95% CI) were calculated by random effects analysis, summary receiver operating characteristic curve was estimated. Sources of heterogeneity were explored by subgroup analysis and bivariate meta-regression.Twenty-six studies were included, of which 19 were included in the meta-analysis (1204 patients). For diaphragmatic excursion, sensitivity was 0.80 (95% CI 0.77–0.83), specificity 0.80 (95% CI 0.75–0.84), area under the summary receiver operating characteristic curve 0.87 and DOR 17.1 (95% CI 10.2–28.6). For the thickening fraction, sensitivity was 0.85 (95% CI 0.82–0.87), specificity 0.75 (95% CI 0.69–0.80), area under the summary receiver operating characteristic curve 0.87 and DOR 17.2 (95% CI 9.16–32.3). There was heterogeneity among the included studies. When performing a subgroup analysis and excluding studies with atypical cutoff values, sensitivity and specificity increased for diaphragmatic thickening fraction; sensitivity increased and specificity decreased for diaphragmatic excursion; when comparing studies using pressure support (PS) versus T-tube, there was no significant difference in sensitivity and specificity; bivariate meta-regression analysis shows that patient position at the time of testing was a factor of heterogeneity in the included studies.Measurement of diaphragmatic excursion and diaphragmatic thickening fraction predict the probability of successful weaning from mechanical ventilation with satisfactory diagnostic accuracy; however, significant heterogeneity was evident in the different included studies. Studies of high methodological quality in specific subgroups of patients in intensive care units are needed to evaluate the role of diaphragmatic ultrasound as a predictor of weaning from mechanical ventilation.The online version contains supplementary material available at 10.1186/s13054-023-04430-9. The process of weaning from mechanical ventilation remains one of the most critical challenges in patients undergoing mechanical ventilation in the intensive care unit (ICU) ; the mulCurrent guidelines recommend several indices applied at the bedside to predict successful weaning from mechanical ventilation. However, they have yet to prove ideal , probablPatients on mechanical ventilation may have a multifactorial deterioration of diaphragmatic function that can lead to weaning failure and prolongation of invasive mechanical ventilation , 9; therThe use of diaphragmatic ultrasound in the intensive care unit is a technique of growing interest due to its portability, speed, and safety. Its use allows reporting on the structural and functional status of the diaphragm and can predict the probability of successful mechanical ventilator weaning . AlthougA systematic review and meta-analysis of observational studies involving intubated patients connected to mechanical ventilation who underwent uni- or bilateral diaphragmatic ultrasound to assess diaphragm function prior to extubation was performed to identify whether there is an association between diaphragm function and extubation success. The systematic review protocol was registered in the Prospective International Register of Systematic Reviews (PROSPERO) CRD42022316349 database, and the systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Two investigators conducted independent systematic literature searches of PUBMED, TRIP, EMBASE, COCHRANE, SCIENCE DIRECT, and LILACS databases published between January 2016 through July 2022, discrepancies between the two investigators were resolved with the intervention of a third investigator. The terms diaphragm, diagnostic imaging, ultrasound, weaning, mechanical ventilation, extubation, ultrasonography, and articles in all languages were included.Methodological quality assessment was performed independently by two investigators using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) instrument, followed by the kappa coefficient to assess inter-investigator agreement; additionally, the certainty of the evidence is evaluated using the GRADE  methodology.The meta-analysis included studies published between 2016 and 2022, and weaning success was defined as maintaining spontaneous breathing for the next 48 h after extubation.Prospective or retrospective observational studies involving adult patients with more than 24 h of invasive mechanical ventilation in whom uni- or bilateral diaphragmatic ultrasound was performed during spontaneous breathing trial.Non-primary studies, studies with less than 20 participants, patients with neuromuscular disease, studies in pregnant patients, case reports, animal studies, and editorials.Rev Man 5.4 the Cochrane Collaboration (2014) software was used for bias assessment, and Metadisc software  for meta-analysis . IndepenSummary receiver operator characteristic curves (SROC) and area under the summary curve (AUSROC) were created to assess the accuracy of DE and DTF for predicting extubation success . Publica2 tests assessed heterogeneity, and the source of heterogeneity was assessed by meta-regression analysis and a subgroup analysis on both sensitivity and specificity. Study characteristics that could cause uncertainty related to the diagnostic accuracy of diaphragm ultrasound were examined; for example, the cutoff values used as a reference, the risk of bias assessed concerning index test and flow and times, the type of spontaneous breathing trial (pressure support (PS) or T-piece), the homogeneity versus heterogeneity of age, the prevalence of success, and the position of the patient. In addition, an analysis was performed to identify whether the cause of the requirement for mechanical ventilation could influence the diagnostic performance of ultrasound.The Cochrane Q and IIn the initial search, 2845 articles were obtained in six databases, 85 duplicates and 2700 articles were eliminated by titles and abstracts, leaving 60 articles; subsequently, the full-text reading proceeded, eliminating 34 articles.  , 25, 34,The results of the risk of bias assessment for the studies included in the meta-analysis are shown in Fig. In most studies, diaphragmatic ultrasonography was performed during the spontaneous breathing trial with a variation of the exact timing of the test, and in other studies, diaphragmatic ultrasonography was performed before and after extubation. Most studies did not clearly report the elapsed time between diaphragmatic ultrasound and extubation , 33, 34.The method of patient selection was not reported in most studies. Several studies did not present the flowchart explaining in detail the patient selection and follow-up , 35, 37.The outcome of weaning varied according to the definition of extubation success or failure and, in some studies, needed to be clearly defined. In most of the included studies, the index test (ultrasound of the diaphragm) was interpreted without knowing the outcome of weaning.No publication bias is evident for the studies that analyzed DE, funnel plot see Fig. , and EggNineteen studies were included in the meta-analysis , 36, 37,Figure 2 65.1% Chi-square 45.86 P 0.001; I2 67.8% Chi-square 49.7 P 0.001, respectively) and for DTF ; the threshold effect measure was evaluated by Spearman correlation, obtaining a value of 0.125 (P 0.63) for DE and − 0.198 (P 0.47) for DTF.Additional file A Fagan nomogram was constructed to illustrate diaphragm ultrasonography's pre- and post-test probability of predicting extubation success Fig. . The pre2 68.5% Chi2 34.9 P 0.002) with no changes in heterogeneity; specificity increased to 0.78 (0.72–0.83 I2 56.9% Chi2 25.5 P 0.01), with decreasing heterogeneity, and AUSROC increased to 0.90; continuing with the manual analysis by subgroups, studies with atypical values of sensitivity [2 43.9%, Chi2 23.1 P 0.03); and decreased specificity to 0.77 (95% CI 0.71–0.83); likewise, decreased heterogeneity ; the AUSROC increased to 0.88  and specificity to 0.77 (I2 60% Chi2 27.7 P 0.003) without significant changes in the heterogeneity for DTF; concerning DE, there is an increase in sensitivity to 0.82 (I2 60% chi2 40 P 0.003) with no change in heterogeneity, no change in specificity for diaphragmatic excursion; the AUSROC was modified to 0.88 for a diaphragmatic excursion and 0.89 for DTF ; it was evidenced that patient position was a cause of heterogeneity for the diagnostic accuracy of the diaphragmatic thickening fraction in the studies included in the meta-analysis  (see Additional file The results of this study suggest adequate accuracy of diaphragmatic ultrasound in predicting weaning success; the combined sensitivity and specificity of DE and the AUSROC were 0.85, 0.75, 0.87, respectively, and for DTF 0.80, 0.80, 0.87. Our data show a satisfactory diagnostic accuracy for predicting extubation success. It is relevant to report that there was heterogeneity in the sensitivity and specificity of the studies included in the meta-analysis; likewise, several studies presented significant methodological weaknesses, two studies with a high risk of applicability in patient selection, and several studies with unclear risk of bias in patient selection, flow and timing.During the last few years, some systematic reviews and meta-analyses have been published on the usefulness of diaphragmatic ultrasound in predicting the success or failure of weaning in patients undergoing mechanical ventilation –41. The This research included five new studies compared to the previous meta-analysis  that maiThe high sensitivity values reported in the present study indicate that patients with values above approximately 29% for DTF and > 1 cm for DE have a high probability of successful extubation; however, it is essential to mention that weaning success may be influenced by additional factors such as nutritional status, respiratory and cardiovascular integrity and psychological conditions to mention a few .We can evidence in the limitations of this study; the possible biases that may contain each of the studies included in the meta-analysis as randomized trials were not included; the absence of a common reference value for diaphragmatic thickening and excursion fraction may introduce biases that cause measurement imprecision; sex was not considered in the analysis by subgroups, nor the time on mechanical ventilation before the spontaneous breathing trial and ultrasound measurement; and these could influence the ultrasound result.In contrast to the findings of this meta-analysis, the two studies mentioned here found no association between values below the cutoff point of diaphragmatic excursion and thickening fraction measured with ultrasound and the outcome of weaning from mechanical ventilation , 26. MarSlight variations in the measurement between observers may affect the measurement result and cause heterogeneity; it is undoubtedly an observer-dependent technique; despite this, several studies have concluded that diaphragmatic ultrasound measurements are reproducible.The results of this study have implications for clinical practice, showing that diaphragmatic ultrasound is a technique that can be used in the intensive care unit during the spontaneous breathing trial to contribute to objectively predict the success of weaning from mechanical ventilation; it is a portable, fast, noninvasive, simple and safe technique that does not emit any ionizing radiation that affects health-care personnel; However, given the high heterogeneity found, which is frequent in meta-analyses of diagnostic tests, the results of the pooled measurements should be interpreted with caution, especially in the different subgroups of critically ill patients, in order to achieve a personalized determination of the optimal result.Therefore, access to diaphragmatic ultrasound in the intensive care unit should be generalized, and priority should be given to achieving its universal use, especially in routine respiratory monitoring, to guide the management of patients undergoing mechanical ventilation.The results of this systematic review and meta-analysis show that measurement of diaphragmatic excursion and diaphragmatic thickening fraction predict the probability of successful weaning from mechanical ventilation with satisfactory diagnostic accuracy; however, significant heterogeneity was evident in the different included studies. Studies of high methodological quality in specific subgroups in intensive care unit patients are needed to evaluate the role of diaphragmatic ultrasound as a predictor of weaning from mechanical ventilation.Additional file 1. Search strategy, supplemental Tables, and supplemental Figures.


All taxa are characteristic of both deciduous and evergreen Mediterranean vegetation, and this data might indicate that the gathering of woods employed for the human cremation/s occurred either on site, or in its vicinity. However, considering both the large distribution of the identified taxa and data about human mobility, it is not possible to conclusively determine the origin of the wood used in the cremation(s). Chemometric analysis were carried out to estimate the absolute burning temperature of woods employed for the human cremation/s. An in-lab charcoal reference collection was created by burning sound wood samples of the three main taxa identified from Pit 16, Olea europaea var. sylvestris, Quercus suber (evergreen type) and Pinus pinaster, at temperatures between 350 and 600 °C. The archaeological charcoal samples and the charcoal reference collection were chemically characterized by using mid-infrared (MIR) spectroscopy in the 1800–400 cm-1 range, and Partial Least Squares (PLS) regression method was used to build calibration models to predict the absolute combustion temperature of the archaeological woods. Results showed successful PLS forecasting of burn temperature for each taxon (significant (P <0.05) cross validation coefficients). The anthracological and chemometric analysis evidenced differences between the taxa coming from the two stratigraphic units within the Pit, SUs 72 and 74, suggesting that they may come from two different pyres or two different depositional moments.Anthracological analyses of charcoal samples retrieved from Pit 16 of Perdigões , a secondary deposition of cremated human remains dated back to the middle of the 3 Perdigões is a large set of ditched enclosures, nowadays classified as a National Monument, located in Reguengos de Monsaraz municipality, Évora district, in the inner Alentejo region, centre-southern Portugal . It holdrd BC), located in the central area of the enclosures [Interpreted as a ceremonial and aggregation centre, these ditched enclosures have several funerary structures, revealing diversified funerary practices in terms of architecture, location within the site, body treatment and votive materials –3. The aclosures , 3. SeveIn southern Portugal and more generally throughout the Iberian Peninsula, there are no other Chalcolithic funerary contexts with the characteristics of Pit 16. Burial rituals in this area during this period were quite heterogenous, including dolmens, tholoi, pits, ditches and hypogea , 5. CremPerdigões, along with other recent archaeological discoveries, led to a revision of the Megalithic literature , 2, 4–11Overall there is a general lack of studies of funerary charcoals across Europe, with only a few studies published about Prehistoric sites –27 with In the literature a charcoal assemblage deriving from human cremation deposits is considered a single or a short time span episode . The maiThe aims of this study are:taxa from Pit 16.To characterize the different wood taxa identified from the Pit.To evaluate the absolute burn temperature of the tree main wood To obtain information regarding this unusual Chalcolithic funerary context, exploring the existence of one or different moments of secondary deposition of human bodies.Two different approaches have been used to achieve these goals. The first one includes the anthracological analysis of the charcoal assemblage, and the second one comprises the FT-MIR/chemometric analyses of archaeological charcoals and sound wood samples (of the same species identified) combusted at different temperature .We consider this approach a good option to successfully evaluate the charcoal assemblage from Pit 16. Results were useful for a better understanding of the archaeological context and the related ritual/s.The partial combustion process of wood either in a forest fire, in a bonfire or in a funerary pyre is well known. It normally produces charred material which contains a significant amount of refractory carbon (C) , 43. ThiThe concentration of aromatic compounds within the chemical structure of the charcoal particles is significantly associated with direct and indirect factors. Direct factors are linked to combustion characteristics, including temperature, fire permanence and heating rate (°C/min). On the other hand, indirect factors are connected to the fuel (wood) properties, like the type of wood, size, wood resilience and moisture –49. ThusCurrently, different techniques are available for the charred organic matter evaluation of complex matrices such as soils and sediments, as well as to assess their burning temperatures. One of these techniques is the reflectance, which is considered a valuable tool to estimate the burn temperatures of modern and archaeological charcoal using a Reflected Light Microscope , 53. AnoThe combination of FT-MIR spectroscopy and multivariate analytical techniques, such as Partial Least Squared (PLS) regression , has demPit 16 is interpreted as a secondary funerary collective deposition, a singular funerary context amongst the Iberian Chalcolithic. The pit contained human remains of at least 9 individuals (6 adults and 3 sub-adults), mixed with other burnt artefacts  as well as a large number of charcoals. The pit is circular, with a profile shrinking at the top, presenting a diameter of 0.92 m at the top and 1.50 m at the base, being 0.90 m deep . In totaQuercus suber, Quercus rotundifolia, Quercus coccifera), Arbutus unedo, some Pinus spp. and Olea europaea var. sylvestris, along with many sclerophyll shrubs, like Cistus spp., Rosmarinus officinalis, Lavandula spp., Myrtus communis, Pistacia lentiscus or Phillyrea sp.; some deciduous trees are also present near streams and other watercourses, like Fraxinus angustifolia, Populus sp., Salix sp..Nowadays, in natural areas, the woody vegetation cover is made up mostly of evergreen oaks  were additionally treated and observed in the laboratory using a stereo-zoom microscope (model LEICA M205C equipped with a camera) to remove sand grains, small ceramic, and animal bone remains and to isolate charcoal fragments  to proceed with the anthracological analysis. A total of 754 charcoals were recovered.The charcoal fragments were manually fractured to obtain the three anatomical sections  under a stereo-zoom microscope (LEICA M205C equipped with a camera). The charcoal’s anatomical features were determined using a reflected light optical microscope (LEICA DM2500 equipped with a camera) at different magnifications (50–1000x). A selection of charcoal fragments was analysed with a Scanning Electron Microscope (SEM) to obtain high-resolution images. Wood atlases –65 were Quercus ilex L.), kermes oak (Quercus coccifera L.) and cork oak (Quercus suber L.) [With respect to the evergreen oak’s wood samples identification, it is significant to reiterate that it was not possible to make any distinction between the evergreen oak tree species. In Portugal, nowadays, as well as in the Mediterranean region, this group includes holm oak (uber L.) , 75.Olea europaea var. europaea) is not differentiable, at the anatomical level, from the wild one (Olea europaea var. sylvestris). However, studies concerning the beginning of the cultivation of this plant in the south of the Iberian Peninsula suggest that it started only in the Roman period [Olea from Pit 16 could probably be ascribed to the wild variety, O. europaea var. sylvestris.Regarding the identification of the olive wood samples, the cultivated species  and the mean values (%U) obtained using the “ubiquity correction” method [taxon  on n different stratigraphic units (SU) is calculated as: %U = %A (SU1) + %A (SU2) + %A (SUn) / n. This method performs a correction for ubiquity of the relative frequencies of each taxon. Charcoal size classes  for each wood taxon identified were also collected.For data interpretation, ” method . The meataxa identified from Pit 16 with the anthracological analysis, namely olive, evergreen oak, and maritime pine woods have been sampled in the proximity of the archaeological site , wooden samples belonging to the wild variety, Quercus suber have been chosen as reference material, considering the abundance of this species in the Alentejo region [3, ≈ 3 g) using an automatic saw, then washed with distilled water and dried for 24 h. Samples were then positioned into ceramic crucibles, heated in the absence of oxygen, between 350 and 600°C, the typical burning temperature of wood in natural fires [For the evergreen oaks, samples belonging to o region . After sal fires . In totaSamples remained within furnace during 90 min at maximum temperature. For each heating step, two replicates of each taxon were burnt. At the end of the heating period, a sample of each laboratory-based combusted charcoal was photographed by SEM after the gold coating procedure.taxon were powdered using an agata mortar and combined to create one composite sample for each different wood taxon.An aliquot from the two charred replicas of each modern wood sample was selected for Fourier Transform Mid-Infrared analysis. The two charred aliquots of each Olea europaea, Quercus spp. (evergreen) and Pinus pinaster were detached for FT-MIR analysis. As for modern samples, the two aliquots of each archaeological taxon were powdered and then combined to create one composite sample to be analysed.Two aliquots of each archaeological sample of ™ Spectrometer , with Attenuated Total Reflectance (ATR) module , at a wavelength range of 4000–400 cm-1 and a resolution of 2 cm-1. To improve the signal to noise ratio, 60 spectra were co-added and averaged for each recorded spectrum. Spectral data were background corrected to a reference spectrum obtained prior to every measurement, and some spurious absorptions, such as peaks from atmospheric CO2, could be removed.FT-MIR spectra were obtained using an Alpha -R-1 . The spectral pre-processing treatments consisted of a light scatter and baseline correction by Standard Noise Variate (SNV), de-noising through a median filter, and mean centering [Partial Least Squares (PLS) regression models were generated using the ParLeS software . This meentering . This prentering , 91, 92 entering . The Rooentering , 58.taxa were determined at the species level, Olea europaea, Pinus pinaster, Fraxinus cf. angustifolia, Arbutus unedo, at the section level, Quercus spp. (evergreen), at the genus level, Cistus sp., and at the family level, Fabaceae. Few charcoal fragments were identified only at the division level, Angiosperm , evergreen oak tree (Quercus spp. (evergreen)), and maritime pine tree (P. pinaster)  and evergreen oak . Finally, the maritime pine is the third taxon in abundance, accounting the 20.7% and the 29%U of the totality  . The mostotality , Fig 6A.dominant , Fig 6B.taxa distribution within the samples , samples from both SUs 74 and 72 showed the presence of vitrification. This feature was observed in 85 (37.2%) fragments of this taxon. For the P. pinaster samples from SU 72, fractures in radial directions  were recorded for a total of 90 (90%) charcoal fragments. Moreover, for 27 charcoal fragments of this species, radial groves in the inner part of tracheids were recorded.Regarding charcoal fragments of With reference to charcoal size, the olive tree is the species with the highest quantity of the largest charcoal, with 71 fragments with values between 1 and 1.99 cm and 9 fragments between 2 and 2.99 cm., followed by the maritime pine and by the evergreen oak, for which the largest charcoals collected are represented by only 14 fragments .taxa it is represented by few fragments of narrow-leafed ash wood (F. cf. angustifolia). This taxon was recorded only in the SU74, and its presence does not exceed the 1.3% and the 0,9%U (Cistus sp.) legume wood (Fabaceae) and strawberry tree wood (A. unedo), accounting for the 8.4% and the 7,4%U of the total , identified on the genus level as Cistus sp.  was identified in SU74. It was not possible to identify the Angiosperm charcoal fragments beyond the division level due to their state of conservation.The remainder of arboreal he 0,9%U , Fig 5E.he total . The mosstus sp. . Betweenain taxa . Finally-1, of the archaeological charcoal samples from SU74 , successfully predicted the crematory temperature of each taxon. Comparison with an alternative model derived from the fully randomised dependent variable that had no connection (p > 0.05) with infrared data validated the model’s accuracy. The PLS model of Olea showed the highest significant prediction , following by Quercus and Pinus . In all cases, the RMSE and AIC values suggested the use of four factors for all taxa. taxa used to obtain the different PLS models. The superposition of the average infrared FT-MIR spectrum of each taxon with the VIP trace indicates the best diagnostic bands to predict the combustion temperature of each taxon. The most significant FT-MIR bands to predict the burning temperature in the case of Olea model were those positioned at 1730, 1580, 1410, 800 and 740 cm-1, while for the Quercus model, the predominant bands were located at 1580, 1410, 1370, 800 and 740 cm-1. In the case of Pinus model, the main bands were identified at 1730, 1410, 1370, 1245 and 1040 cm-1.The cross-validation plots  for the burning temperature of each set of modern wood charred Olea samples, the statistic model used the bands at 1730 and 1580 cm-1 corresponding to vibrating C = C and C = O bonds, respectively, in the aromatic rings [-1, related to C–C stretch (aromatic ring) of lignin compounds [-1, linked to methyl C–H rock in alkene compounds [Olea. This taxon also used the bands at 800 and 740 cm-1 which are typical of polysaccharide compounds [Quercus PLS model used the same FT-MIR bands as the model of Olea; however, in this case, the band at 1730 cm-1 was not determinant. On the other hand, the model of Pinus employed the band at 1245 cm-1 linked to acidic compounds of the resin [-1, which indicate the presence of resinous compounds [-1, that is related to the C–O stretch, displayed a noticeable high VIP value.Each forecasting model was made up of characteristic infrared bands . In the ic rings . In addiompounds  and 1370ompounds  were alsompounds . The Quehe resin . This atompounds . In additaxa. In SU74, the Pinus pinaster showed the highest combustion temperature (576 ± 24°C) in comparison to Olea europaea (544 ± 20°C) and Quercus spp. (evergreen) (509 ± 6°C). The one-way ANOVA analysis showed that temperatures were statistically significant  among different wood taxa (p< 0.05) highest crematory temperature is registered by O. europaea (506 ± 12°C), while Quercus spp. (evergreen) and P. pinaster  showed no significant (p>0.05) differences among them.The predicted absolute burn temperature for the archaeological charcoal samples identified in SU74 and SU72 are displayed in ood taxa . Howeverood taxa , the sigAll taxa identified are characteristic of the Mediterranean pine forest and sub-Mediterranean sclerophyllous vegetation .taxa in the anthracological record of Pit 16: O. europaea, Quercus spp. (evergreen) and P. pinaster  and P. pinaster. There are plenty of recorded instances of use of ash wood for human archaeological cremations, although its use seems to become consistent rather than sporadic only during the historical time [taxa of Cistus sp., A. unedo and Fabaceae, numerous studies have already widely attested the use of shrubs and small trees in human cremation rituals. The Cistus sp. is a small perennial shrub that grows in dry and rocky soils of the Mediterranean area and its presence has been observed in other archaeological human cremations, especially during the Roman period [A. unedo tree wood in human cremations [A. unedo, whose presence Pit 16 at Perdigões is limited only to SU 74 , P. pinaster and F. cf. angustifolia , for the made up of the pyre/s formed by charcoal of the pit.P. pinaster could be the result of a conspicuous loss of humidity on burned fresh wood [P. pinaster wood shortly after the cutting of the tree.The radial cracks observed in some charcoals of esh wood ; althougesh wood . The absesh wood  on charcP. pinaster, namely grooves inside tracheids visible on the longitudinal sections of charcoals, suggests a concentric growth in this part of the tree, developed during its life to prevent fall under its own weight [The reaction wood feature observed on some charcoal fragments of n weight .The characteristics and properties of the identified woods provide information regarding the selection, intentional or not, and use of wood by the Chalcolithic community which prepared the pyre/s deposited in Pit 16.taxa. Nowadays the burning properties of olive wood are well appreciated, in particular due to its long-lasting fire and the pleasant aroma which derives from its burning. Furthermore, its sacredness is emphasized in the ethnographic literature [taxa of the anthracological record, this wood might have been intentionally selected by the community who made the ritual, and the selection could have been made for cultural and/or social reasons. As for the oak wood, the literature underlines how it also provides an excellent fuel, long lasting and with high calorific power [Let us first discuss the three main terature . It shouic power –117. On ic power , 118. Thic power , 108.Ash wood produces an excellent fuel, and its property of burning, both when is seasoned than when is fresh, is well known. The shrubs identified in the anthracological record of Pit 16, due to small-calibre woods, would seem particularly useful in lighting fires. Moreover, they emanate a pleasant smell during their combustion, and their use in these type of contexts may represent an anthropic symbolic choice . The pretaxa, if appropriately compared with the results of pollen analysis at Perdigões [genera observed in the anthracological record of Pit 16, i.e., Olea, Pinus and Quercus. Inside the pollen records, the presence of various species belonging to Cistus and the presence of Fabaceae species has also been verified [Quercus and Oleaceae, the olive family to which belong both Olea and Fraxinus genera, were identified [Assemblages of concentrated charcoal, especially those coming from a single context, cannot be considered reliable for the reconstruction of the vegetation surrounding the archaeological site , 30, 35.erdigões , 122 andverified . In the entified , 124. Acentified . These dentified , 126, whentified , 128.taxa collected in the secondary cremation of Pit 16. At this stage it is not possible to exclude that the primary cremation occurred neither in the vicinity of the archaeological site nor in slightly more distant areas.Based on this data, it is challenging to formulate a single hypothesis concerning the origin of the wood taxa within the two stratigraphic units (SU72 and SU74)  , Fig 6B,) . This result was in line with data reported from other authors  who detep<0.05) difference among the temperature reached by the different types of wood. This difference may be due to different factors, such as: i) the chemical composition of each type of wood (inflammable compounds like resins or epicuticular waxes); ii) the wood moisture content [taxon.The burn temperature calculated by the combination of FT-MIR and chemometrics analysis, in this experiment, may suggest the existence of a single cremation for SU 74 . On the other hand, the statistical analysis of the combustion temperature of charcoal sample particles found in SU74 determined the existence of a significant . In the case of Pinus samples, a significant temperature difference of 89°C , burning at lower temperatures. The possible use of some green woods for the cremation would explain the low combustion temperature of Pinus samples from SU 72. In contrast, in SU 74, the highest temperature reached by Pinus may reflect the use of seasoned individuals with a relatively noticeable amount of resinous material (high degree of flammability). Therefore, in both cases, the wood seasoning of pine samples could be considered an important actor to determine the temperature of combustion.In the case of SU72, there were not significant differences between the burning temperature of  of 89°C  was obsetaxa of Pit 16, especially regarding charcoals fragments of P. pinaster, observed between the two stratigraphic units SUs 72 and 74, we can put forward the hypothesis of the existence of 2 different depositional moments, inside the Pit and consequently coming from two different primary rituals (cremations). This hypothesis was already raised with the anthracological analysis and it corroborates the archaeological data regarding the distinction of the two different stratigraphic units.From the combustion temperature differences of the tree main Quercus charcoals in SU72 is comparable to that one reached in SU74. This may be due to the use of Quercus suber wood in both crematory events. The existence of a burning protective cover, like suberin, on trunk would have been able to limit and “average” the combustion temperature of the Quercus wood in both crematory events. Moreover, Q. suber tree displays a relatively high abundance of this species in the Alentejo region [The absolute relative temperature reached by o region . Resultso region  demonstrThe anthracological study of Pit 16 has proved to be particularly relevant for the understanding of human choices of timber for funerary purposes, demonstrating its importance in discerning the complexity of the human cremation process: do the culturally determined wood selection and the wood selection based on the territorial availability represent two different solutions, two coexistent solutions or are they intrinsic solutions?If we consider the parameter known as the "principle of least effort" , we mustAnswering such a wide question by looking for a singular solution, when human choices were dictated by multiple factors in all periods of communal human existence, past and present, is an unproductive effort.taxa are O. europaea, Quercus spp. (evergreen) and P. pinaster. All the species identified in the study are characteristic of both deciduous and evergreen Mediterranean vegetation. The origin of plants selected seems to indicate the occurring of the human cremation/s either on site, in the vicinity or in an area with access to a similar vegetation.The charcoal record of Pit 16 is a mixture of different trees species. The most abundant tree Regarding the investigation of the charcoals burn temperature, this work has demonstrated the validity of the combination of FT-MIR and chemometric analyses to establish the absolute combustion temperature of archaeological charcoals from a human cremation. Considering both the data from chemometric and anthracological analyses, it was possible to speculate the existence of two different depositional moments inside Pit 16.As for the understanding of human choices of wood species employed for funerary purposes, results from this study seem to indicate that both cultural choices and the ones dictated by the environment might have driven the Chalcolithic community of Pit 16 in the wood selection for the funerary ritual/s.This study demonstrated the validity of the use of different approaches, an archaeobotanical, a chemical and a statistical approach, to shed light on the role of woods within the ritual of cremation within the funerary practices present in Perdigões during the Chalcolithic.S1 Data(XLSX)Click here for additional data file.


Fragaria × ananassa) is among the most widely cultivated fruits with good taste and rich nutrients. Many strawberry species, including white strawberries, are planted all over the world. The metabolic profiles of strawberry and distinctions among different cultivars are not fully understood. In this study, non-targeted metabolomics based on UHPLC-Q-Exactive Orbitrap MS was used to analysis the metabolites in 10 strawberry species. A total of 142 compounds were identified and were divided into six categories. Tochiotome may differ most from the white strawberry (Baiyu) by screening 72 differential metabolites. Histidine, apigenin, cyanidin 3-glucoside and peonidin 3-glucoside had potential as biomarkers for distinguishing Baiyu and another 11 strawberry groups. Amino acid metabolisms, anthocyanin biosynthesis and flavonoid biosynthesis pathways were mainly involved in the determination of the nutrition distinctions. This research contributes to the determination of the nutrition and health benefits of different strawberry species.Strawberry ( Fragaria × ananassa) is among the most widely cultivated fruits, with a total of approximately 8,882,500 tons production all over the world in 2019 [Strawberry , Fenyu (FY), Guangdian (GD), Benihoppe , Ssanta (SDH), Kaorino (SZ), Tongzhougongzhu (TZGZ), Yuexiu (YX), Tochiotome (YY), and Akihiime (ZJ). In particular, since the Benihoppe cultivar was widely cultivated in China, a total of three groups  of this cultivar from the same season and region were chosen in this study. The strawberries were collected from Beijing (China) during February and March 2022. The samples were immediately frozen in liquid nitrogen after harvesting and stored at −80 °C until the extraction of metabolites.Methanol and acetonitrile (liquid-chromatography-grade) were purchased from Merck . Deionized water (18.2 MΩ) was obtained from a Milli-Q water purification system . Formic acid and ammonium formate were supplied by Sigma-Aldrich .v/v) was added to the samples. After that, the mixtures were vortexed for 1 min and subjected to ultrasonic vibration for 30 min. They were then centrifuged for 5 min at 10,000 r min−1 at 4 °C. The supernatants were collected carefully and filtered through 0.2 µm polytetrafluoroethylene filters for analysis. Four replicates were prepared in each strawberry group. Equal quantities of the sample supernatants were mixed to prepare the quality control (QC) sample, which was embedded in the batch of every six samples during the analysis to assess the system’s stability.Frozen strawberry samples (1 g) were placed into 15 mL Eppendorf tubes and pre-chilled in liquid nitrogen. Then, 10 mL methanol-water  equipped with an Xbridge Amide column  at 40 °C. The flow rate was 0.3 mL minm/z.Mass spectrometric detection was performed on a Q-Exactive Orbitrap mass spectrometer equipped with an ESI ion source operated in the positive and negative ion modes, respectively . The experimental parameters were as follows: spray voltage, 3.00 kV; sheath gas pressure, 30 psi; auxiliary gas pressure, 10 arbitrary units; capillary temperature, 320 °C; auxiliary gas heater temperature, 350 °C; scan mode, full MS -dd/MS2 ; and scan range, 70–1050 https://pubchem.ncbi.nlm.nih.gov/ (accessed on 1 May 2022) according to the MS2 information. Differential metabolites were screened out based on log2 Fold Change > 1 and p-value < 0.05. The information is further expressed as volcano plots in the https://www.metaboanalyst.ca/ (accessed on 30 June 2022).Non-targeted metabolomics data were imported to the Compound Discoverer 3.3  software for analysis, including peak filtering, alignment, identification, and normalization. Relevant parameters were set as follows. The retention time tolerance was 0.2 min, and the mass tolerance was 5 ppm. The signal-to-noise ratio was 3, and the minimum peak intensity was 1,000,000. The intensity tolerance was set at 30%. The results of compound identifications were further confirmed by PubChem database  were detected. All of the peaks were finally confirmed to 113 compounds (positive) and 37 compounds (negative) by the fragment information in the Pubchem library. Furthermore, eight of the identified compounds were detected in both the positive and negative ion modes. These were glutamic acid, histidine, apigenin, cytidine, phenylalanine, aspartic acid, asparagine, and O-acetylserine. Therefore, a total of 142 compounds were finally identified. Detailed information of these compounds are shown in Our results confirmed that small molecule metabolites such as organic acids, amino acids, vitamins and polyphenols were widely detected in strawberries. Additionally, the metabolite contents varied in different strawberry cultivars. In this study, a total of eighteen organic acids were confirmed to exist in different strawberry cultivars, such as citric acid, malic acid, quinic acid and mevalonic acid. The results were consistent with previous studies of fresh strawberry samples . Kinds oIn our study, a total of 18 kinds of polyphenols were identified, including cyanidin 3-glucoside, peonidin 3-glucoside, cyanidin, tiliroside, et al. As can be seen from Amino acids were widely detected in various strawberry cultivars in the present study. This was in line with the profiled amino acids in strawberry juice in a previous study . The ricBesides the metabolites of the strawberry, various kinds of exogenous compounds were detected at the same time, such as chlormequat and difenoconazole. These compounds are presumed to be used during the strawberry growing process. For example, chlormequat was one of the pesticides in strawberry with the highest detection percentages, according to a previous study . Similar2 > 0.995 and Q2 > 0.988) in 2 and Q2 (the two right-most points) were always greater than the transposed values on the left (the left scatter points). In addition, the intercepts of Q2 were less than zero in all of the OPLS-DA models. These results demonstrated that all of the established OPLS-DA models were reliable and not overfit. A table of the Std. dev. values in all of the groups has been provided in For better understanding of the distinctions between the endogenous metabolites among different strawberry cultivars, multivariate statistical analysis was conducted. 2Y (the explanatory ability of the model to classification variable Y) and Q2 (the predictability of the model) obtained after cross-validation were used to evaluate the effectiveness of the model. Finally, the replacement test was passed (permutation test), the order of Y was changed several times to obtain different random Q2 values, and the validity of the model was further tested  in all of the pair-wise comparisons. Taking a further look at the contents of these four potential biomarkers, they all presented higher contents in other strawberry cultivars than that in the BY group. These results suggest the differences in metabolite contents of various strawberry species, and the distinctions further determined the differentiation of taste, nutrition and even color between the BY species and other strawberry species. Based on the principles of Histidine is a semi-essential amino acid for humans and other mammals. It has important functions in mood disturbance and response time . During Apigenin is a kind of flavonoid with potent anti-oxidant effect. Apigenin and its various derivatives are abundant in strawberry, especially in wild strawberry, such as the native Chilean red strawberry ,31. In tCyanidin 3-glucoside and peonidin 3-glucoside are two kinds of anthocyanins with significant content differences between Baiyu and other strawberry cultivars. It is well known that cyanidin 3-glucoside was one of the major anthocyanins in strawberry. However, peonidin 3-glucoside was first identified in strawberry in 2010 . They plAccording to the 16 differential endogenous metabolites that were screened out multiple times (10 and 11 times) in comparison with BY, the main metabolic pathways involved are summarized in In the present study, we conducted a comprehensive metabolomics analysis of the metabolites in strawberry and compared the differences in their contents in 10 different cultivars. A total of 5011 peaks were detected in the positive and negative modes. They were finally identified as 142 compounds, and were further divided into six categories. The results showed that small molecule metabolites such as organic acids, amino acids, vitamins and polyphenols were widely detected in strawberries, and their contents varied with different strawberry cultivars. Multivariate analysis revealed the distinctions between the white strawberry cultivar (Baiyu) and other strawberries. Histidine, apigenin, cyanidin 3-glucoside and peonidin 3-glucoside had potential as biomarkers for distinguishing Baiyu and the other 11 strawberry groups. Pathway analysis results showed that the main metabolic pathways based on the occurrence of multiple differential metabolites were related to amino acid metabolisms, flavonoid biosynthesis and anthocyanin biosynthesis. These findings suggested that the distinctions between BY and other strawberry cultivars were mainly reflected in the differences of amino acids and polyphenols. The obtained results revealed the potential nutritional biomarkers among the ten strawberry varieties and increased our understanding of the association between strawberry cultivars and metabolite differences. Overall, the present work contributes to a scientific basis for improving strawberry nutrition in breeding and cultivation.


Findings reveal that the infill percentage significantly influences the i/CpV ratio for tensile, compression, and hardness tests, while flexural tests are influenced by layer thickness. Impact strength is influenced nearly equally by both factors, with material-specific variations. The desirability function proved useful for optimizing processes with multiple responses, identifying the optimal parameters for the FDM process: a layer thickness of 0.15 mm with 100% infill percentage for PLA, a layer thickness of 0.20 mm with 100% infill percentage for annealed PLA, and a layer thickness of 0.15 mm with 100% infill percentage for ABS. Overall, this study guides efficient 3D printing parameter selection through a technical-economic optimization based on value analysis.This article introduces a multi-objective optimization approach for determining the best 3D printing parameters (layer thickness and infill percentage) to efficiently produce PLA and ABS parts, extensively analyzing mechanical behavior under tests for different traits such as tensile strength, compression, flexural, impact, and hardness. The value analysis method is used to optimize settings that balance use value ( In the contemporary landscape of additive manufacturing, fused deposition modeling (FDM) has emerged as a prominent technique, offering novel avenues for design innovation and production efficiency. Out of all the available 3D printing methods, FDM stands out as an economical and fast printing technique . HoweverWhile FDM’s capacity for intricate design realization is widely acknowledged, its optimal implementation necessitates a holistic understanding of the multifaceted parameters governing the manufacturing workflow. Moreover, the intricate amalgamation of material utilization, production duration, and mechanical performance within the FDM framework underscores the significance of pioneering strategies that harmonize technical finesse with cost-conscious practices. The efficacy of 3D printing, alongside the mechanical characteristics of the end component, hinge on a spectrum of printing parameters including layer thickness, printing speed, infill density, filling pattern, printing material, and various other factors . The preThe study detailed in  investigMaguluri et al.  exploredThe research in  investigIn , the appA similar study was performed in , with thVishwas et al.  focused Many other studies ,17,18,19Sustainable manufacturing aims to integrate quality, environmental consequences, and cost implications, leading to a shift in the focus of optimization objectives . TherefoRefocusing on the relationship between production costs and printed component production, the current emphasis  lies on The study in  used staThe work detailed in  extensivThe investigation performed in  found thIn specialized works, both the term “Value Engineering” and “Value Analysis” are used, both reflecting the same content but at different stages of the product’s existence . Value eiV) and the direct and indirect production costs it generates (pC).Starting from social needs and based on the latest advancements in science and technology, value engineering/analysis studies aim to establish an optimal relationship between the use value of the analyzed product  ViCpi/CpV ratio can be increased through , eC is the cost of energy [Euro], To calculate the production cost, we used the relation :(2)Cp=CmpaC was calculated with the formula:pC is the production cost calculated with Formula (1) for the as-built samples, aT is the annealing time [h], enaC is the energy consumption of the annealing device [kW], and sn is the number of samples annealed simultaneously in the oven.For the annealed samples, the production cost enC = 0.35 kW (the energy consumption of the Raise E2 3D printer), mP = 0.022 Euro/g  and Pm (for ABS ), Pen = Euro/kWh , Ta = 3 Testing conditions, such as the shape and dimension of the samples for each experimental investigation performed, are presented in Similar to their impact on mechanical properties, the 3D-printing parameters were observed to significantly influence the production cost due to energy consumption .Although the heat treatment adds supplementary costs to the production, the results from i/CpV increased from 11.65% to 32.29%, based on increasing performance more than cost .The data from i/CpV ratio was obtained at 100% infill percentage and 0.15 mm layer thickness  and 0.20 mm . The ANOVA analysis  revealed that the statistically significant factor influencing the ratio i/CpV for tensile strength is the infill percentage. The same conclusion can be drawn from Pareto charts presented in It was found from iV/pC is notably more influenced by variations in infill percentage.In contrast to the findings reported in , where lCompared with , where eiV (performance) to pC (production cost) decreased by 6.14% to 25.1%. This decrease occurred because the increase in performance was less pronounced than the rise in production costs. Specifically, for annealed specimens, production costs increased between 17.74% and 25.52%, while compressive stress increased by 5.78% to 13.84%. Notably, the only exception was observed in the case of specimens with a 0.1 mm layer thickness and 100% infill percentage, where the compressive stress was 5.88% lower for annealed specimens compared to the as-built ones.The data in i/CpV ratio was obtained at 100% infill percentage and 0.20 mm layer thickness (for as-built PLA and ABS) and 0.15 mm . Also, for all analyzed materials, the ratio i/CpV increased with the increase in infill percentage. The ANOVA analysis and Pareto charts  and at 0.20 mm layer thickness . The ANOVA analysis and Pareto charts  and, respectively, infill percentage —see i/CpV in the case of annealed PLA, while for as-built PLA specimens, the ratio decreases with the increase in infill percentage. For both as-built PLA and ABS materials, the increase in the ratio i/CpV is obtained by increasing layer thickness.In the case of impact strength analysis, both layer thickness and infill percentage influence the iV/pC ratio is visually emphasized in The i/CpV ratio exhibited a decline ranging from 8.34% to 15.57%. This reduction is attributed to the fact that the Shore D hardness values of annealed PLA specimens were consistently lower, decreasing by 5.08% to 12.86%. Concurrently, the production cost increased marginally, by 2.79% to 3.74%.The information from i/CpV is the infill percentage, for all the analyzed materials. For PLA the highest ratio is obtained with 0.15 mm layer thickness and 50% infill percentage, for annealed PLA at 0.20 mm layer thickness and 50% infill percentage and for ABS at 0.20 mm layer thickness and 50% infill percentage.The main effect plots from The objectives of optimization typically involve maximizing, minimizing, or setting target values for a response in order to identify the most favorable processing parameters. In the context of desirability analysis, the objective of the present investigation  was to maximize the response desirability, namely the ratio between the use value and production cost see , simultaDesirability analysis provides values within a range of zero to one, with one indicating the highest level of suitability.D is calculated with the formula [n is the number of responses, id represents the desirability for each individual response, calculated  as in [For the case when the importance is the same for each response, the composite desirability  formula :(6)D=d1⋅y) as in :(7)di=0,iy, iT, iL represent the predicted value, target value, and lowest value, respectively, of the analyzed response of response. To maintain simplicity, the authors treated all responses as equally significant within the optimization procedure  for achieving the highly efficient production of PLA and ABS 3D-printed parts, based on a complex characterization of their main mechanical properties . The investigation used an innovative solution involving value analysis to establish the optimal printing settings that result in maximizing the ratio between the use value  and the production cost of 3D-printed products. In this way, a technical-economical optimization can be performed.✓For the FDM process under investigation, taking into account the mechanical characteristics  and the use value and production cost of specimens made of different materials, the optimum parameters were found.✓i/CpV ratio in the case of tensile, compression, and hardness testing specimens, while for flexural testing specimens, the layer thickness makes the most important contribution. When analyzing impact strength, both layer thickness and infill percentage exhibit nearly equal influence on the i/CpV ratio. However, for PLA and ABS materials, layer thickness holds a slightly greater impact, while for annealed PLA, the infill percentage has a slightly higher influence.The ANOVA analysis demonstrated that the infill percentage is the significant factor with statistical influence on the ✓Annealing has been proven to significantly enhance some mechanical characteristics of PLA 3D-printed parts. In order to obtain a favorable ratio between mechanical performance and production cost, a certain number of samples must be heat-treated simultaneously for an efficient use of energy. The gains in mechanical strength per piece, combined with the efficient use of energy and time during annealing, contribute positively to the cost-effectiveness of the process.✓Prospective investigations within this domain necessitate a targeted assessment of value, centered on the specific applications demanded, with a keen focus on enhancing both utility and production-cost efficiency for industry-specific components. To enhance the rigor of future studies, it would be useful to use precise measurements for factors such as energy consumption and labor costs.Based on the obtained results, the following conclusions can be drawn: