Preparation methods have been found to affect the physical and chemical properties of rice. This study prepared Guichao rice flour with wet, dry, semi-dry, and jet milling techniques. Differences in the particle size distribution of rice flour were investigated in order to assess their impact on pasting, thermal, gel, starch digestibility, and crystalline structure using an X-ray diffractometer (XRD) and a Rapid Visco Analyzer (RVA) across in vitro digestibility experiments. The results showed that semi-dry-milled rice flour (SRF) and wet-milled rice flour (WRF) were similar in damaged starch content, crystalline structure, and gelatinization temperature. However, compared with dry-milled rice flour (DRF) and jet-milled rice flour (JRF), SRF had less damaged starch, a higher absorption enthalpy value, and a higher gelatinization temperature. For starch digestibility, the extended glycemic index (eGI) values of WRF (85.30) and SRF (89.97) were significantly lower than those of DRF (94.47) and JRF (99.27). In general, the physicochemical properties and starch digestibility of WRF and SRF were better than those of DRF and JRF. SRF retained the advantages of WRF while avoiding the high energy consumption, high water consumption, and microbial contamination disadvantages of WRF and was able to produce better rice flour-associated products. Rice is a basic food on which human beings depend for survival and development. Rice has a light taste and a high yield and is easy to store, making it popular with merchants and consumers . About 6During the preparation of rice flour, the milling of raw rice is the most important step, and milling methods mainly include wet, dry, semi-dry, and jet milling. Soaked samples can be wet-milled with water and then dried into rice flour . SoakingBecause of the high starch content in rice flour, starch digestibility has a significant influence on the biochemical indicators of human blood glucose . This maRice flour is the main material used for rice-based foods, and the physicochemical properties and digestibility of raw materials determine food quality, so it is essential to study the preparation of raw materials. This study aims to investigate the impact of four preparation methods on the particle size distribution and its effect on the damaged starch content, pasting, thermal and gel properties, and starch digestibility of flour. This study expects to obtain low GI rice flour with better physicochemical properties that can enhance the quality of rice flour-related products.Guichao rice (early indica rice) was bought from Jiangxi Huadachang Food Industry Co., Ltd.  in 2019. The water content was 8.08 g/100 g, the protein content was 8.36 g/100 g, the lipid content was 0.73 g/100 g, the ash content was 0.77 g/100 g, the total starch content was 84.04 g/100 g, and the amylose content was 21.61 g/100 g. The procured rice was kept at −20 °C before experimental use. The porcine pancreas α-amylase, pepsin, pancreatin, and amyloglucosidase (AMG) were obtained from Sigma-Aldrich Trading Co., Ltd. . The D-Glucose Assay Kit (GOPOD format), Total Starch Assay Kit, Starch Damage Assay Kit, and Amylose Assay Kit were procured from Megazyme International Ireland Ltd. . The chemicals employed in this research were of analytical quality.WRF: In 4 kg of deionized water, 2 kg of sample was soaked at 25 °C for 6–8 h. The soaked rice was mixed with 1 kg of distilled water and coarsely milled in a colloid mill . The coarse pulverized pulp was then milled for 3 cycles with a gap of 0.10–0.15 mm . ThereafSRF: A total of 2 kg of the sample was mixed with the required amount of water and soaked at 25 °C. The sample was sealed and shaken, then left to be tempered at 25 °C. The rice was tempered for 8 h, during which time the rice was shaken every hour to adjust the moisture content to 27%. The rice was then crushed in a cyclone mill  and passed through standard sieves of 80 mesh . The milDRF: A total of 5 kg of rice was milled in a cyclone mill  Co., Ltd., Suzhou, China) and was passed through standard sieves of 80 mesh . The DRFJRF: A total of 5 kg of rice was milled using an airflow ultrafine mill  and was passed through 200 mesh sieve and were stored in a hermetic bag at 4 °C for use .The contents of moisture, crude protein (GB 5009.5-2016), crude fat (GB 5009.6-2016), and ash (GB 5009.4-2016) were determined according to the National Standards of China. The contents of total starch, amylose, and damaged starch were determined using a Total Starch Assay Kit, Amylose/Amylopectin Kit, and Starch Damage Assay Kit , respectively.An X-ray diffractometer  was used to determine the crystalline structure of samples. The determination method was carried out according to the method of Wang et al. (2018) . An apprA Malvern laser particle size analyzer  was used to measure the particle size distribution of the dry powder . D10, D5The pasting properties of the samples were determined using a Rapid Visco Analyzer . Measurements were based on the method of Gao et al. (2019)  with somΔH).A differential scanning calorimeter  was used to determine the thermal properties of a sample. The method of determination was modeled on the method of Li et al. (2017)  with somWhen added to an amount of water, 10 g of rice flour formed a suspension of 15% mass fraction. The determination method was based on the method of Li et al. (2017) . The mixv/v) ethanol to terminate the reaction. In addition, 0.1 mL of glucose (1 mg/mL) was taken as the standard sample, while 0.1 mL of 95% (v/v) ethanol was used as the blank control. Each analysis was repeated three times. Starch digestibility was obtained using the following equation:A is the absorbance per time point, and As (A sample) is the absorbance of the standard. V is the volume of the digestive solution, N is the dilution multiple, and B is the basic dry mass of the sample. The The methods of Syahariza et al. (2013)  and Zou  glucose .tC expresses the glucose concentration at digestive time t, ∞C is the glucose concentration at the final timepoint (240 min), and k is the digestibility coefficient [The starch hydrolysis curves were plotted based on the glucose content of samples sampled at specific time points. The starch hydrolysis rate curves often followed first-order kinetics, as indicated by Equation (2):fficient .AUC) was obtained from the k value, as shown in Equation (3):The area under curve (HI) is the ratio of the sample AUC to the standard sample AUC [The hydrolysis index  [The calculation of . (1992) , is showEach sample was duplicated three times and the average value was obtained. All data were processed using Origin  software and SPSS . The particle size distribution of grain flour affected the physicochemical properties and processing qualities of sample, which further affected the edibility and sensory quality of products . The parThe physicochemical properties and digestibility of sample were largely influenced by the particle size of rice flour . When coDuring rice milling, starch particles can be damaged and even starch molecular structure can be degraded, thus increasing the amount of damaged starch . The maiX-ray diffraction was used as an analytical method to study the crystalline structure of starch. From p > 0.05). The largest difference in crystallinity degree was between SRF and JRF, with a difference of 2.45%, while the smallest difference in crystallinity degree was between DRF and JRF, with a difference of 0.15%. This indicated that the various flour processing techniques had little influence on the crystallinity of rice starch.The crystallinity degree of rice flour obtained under various milling conditions is presented in p < 0.05). The starch molecules were heated and gelatinized to form a viscous gel [p < 0.05), suggesting that the water-holding capacity and network structure of these two flours were improved [Rice flour cannot form a gluten network structure, and the main structure of rice flour products consists mainly of starch gels. Therefore, it is crucial to investigate the effect of various production processes on the pasting properties of sample, which to some extent reflected the quality of products . The rescous gel . On coolcous gel . JRF hadcous gel . Interesimproved ,10. The improved .p < 0.05). However, one study found that samples with smaller particle sizes and more broken starch had lower Tp and Tc values [ΔH reflects the energy of the transition from a crystalline to a non-crystalline structure [ΔH of rice flour between the various milling techniques (p < 0.05). The different milling methods resulted in large differences in damaged starch content. WRF had a maximum ΔH because WRF had less damaged starch and relatively intact granules, thus requiring more energy to break the granule structure and absorbing more heat in the process [ΔH was negatively correlated with damaged starch content, so JRF had the lowest ΔH value of the four samples. In general, the thermal properties of the four samples were associated with changes in particle size and damaged starch content due to preparation methods [When a phase change of the material occurs, it is always accompanied by energy absorption or release. In the DSC analysis, the crystalline structure of starch absorbs heat during the melting process, which appears as a heat absorption peak. The larger the peak area, the more heat is required to melt the crystal and the higher the melting enthalpy change. As shown in c values . This coc values ,39. In ac values . ΔH refltructure . There w process . The ΔH  methods .p < 0.05). Compared to WRF, SRF, DRF, and JRF showed increased resilience, springiness, and chewiness. In terms of hardness, DRF had the highest hardness of 6.96 N, followed by SRF and, finally, WRF and JRF. The gel hardness of WRF was close to that of JRF (p > 0.05). In terms of the adhesiveness index, the viscosity of the other three flours was higher than that of WRF. DRF had the highest hardness, and this was attributed to damaged starch content. Rice flour was easy to setback when broken starch was high. The setback was due to increased adhesiveness caused by the rearrangement of amylose during cooling [The texture of products was influenced by the gel properties of the samples. TPA results are shown in  cooling . High DR cooling ,43. Alth cooling . The aut cooling . Some st cooling ,45. In a cooling .p < 0.05). This may be due to the semi-dry milling method and the wet milling method retaining more intact cell walls in the rice flour. The cell walls reduced the contact area for digestive enzymes and increased the barrier to enzyme entry into the cell [Many kinds of cereals were crushed before production and processing. The degree of milling and the size of the flour not only affected the processing properties, such as water absorption and solubility, but also affected the digestibility of the rice flour products. the cell . The actthe cell . The resThis research compared the effects of dry, semi-dry, wet, and jet milling methods on the particle size, physicochemical properties, and digestibility of rice flour. The results indicate that milling methods had no effect on the crystalline structure of samples. Compared with DRF and JRF, SRF was close to WRF in terms of damaged starch content, crystalline structure, and gelatinization temperature and had a better network structure. DRF had high degree of recrystallisation and the highest gel hardness. In terms of rice flour digestibility, the eGI of SRF was significantly lower than that of DRF and JRF, which was close to that of WRF. In general, WRF and SRF had better physicochemical properties and starch digestibility. Due to the high energy consumption and water consumption of WRF, SRF is more suitable for the preparation of low-eGI processed rice flour products. In future studies, semi-dry milled rice flour will be used to prepare various types of rice flour products with a lower glycemic index, such as rice noodles and rice cakes, and to assess their starch digestibility in vivo and in vitro.


Homo sapiens.Despite the extensive literature on the retrieval of digestible starches from archaeological contexts, there are still significant concerns regarding their genuine origin and durability. Here, we propose a multi-analytical strategy to identify the authenticity of ancient starches retrieved from macrolithic tools excavated at Upper Paleolithic sites in the Pontic steppe. This strategy integrates the morphological discrimination of starches through optical microscopy and scanning electron microscopy with single starch chemo-profiling using Fourier transform infrared imaging and microscopy. We obtained evidence of aging and biomineralization in the use-related starches from Palaeolithic sites, providing a methodology to establish their ancient origin, assess their preservation status, and attempt their identification. The pivotal application of this multidisciplinar approach demonstrates that the macrolithic tools, from which starches were dislodged, were used for food-processing across the Pontic Steppe around 40,000 years ago during the earliest colonization of Eurasia by  In contrast, mechanical processing  of starch-rich plants before consumption are not well-documented across the Late Pleistocene, although these activities may leave behind use-related biogenic residues2 (U-RBRs) on the active surfaces of the pebbles used as ground stone tools (GSTs). Proving the intentional processing of carbohydrate-rich plants through the analysis of potential GSTs is still controversial and often limited by the object biography and possible contamination that may be due to modern starches occurring during activities carried out in the field (excavations), throughout conservation procedures in museum settings, and even in laboratory settings4.The consumption of dietary carbohydrates has been documented since the Middle Pleistocene by isolating starch grains from dental calculi and coprolites5, and structured residues such as pollen, phytoliths, fibers, and starch grains that can be identified through imaging. Among these structured biogenic residues, our focus is on identifying ancient starch grains dislodged from the surfaces of utilized ground stones dating back to 40 ka. We examined samples that came from macrolithic ground stones tools (GSTs) retrieved in three sites across the Pontic Steppe attributed to the early phases of the Upper Paleolithic: Brînzeni I, a cave facing the Prut River basin in Moldova, Surein I, a rock shelter on the southern slopes of Crimea, and from layer III of Kostenki 14-Markina Gora, an open air site along the Don River6. These sites represent three different environmental conditions which might have influenced the conservation of the structured U-RBRs (SU-RBRs).U-RBRs are comprised by two main types of residues: amorphous, lacking recognizable structure8. Morphological identification of starch grains, both modern and ancient, is conventionally conducted using optical microscopy (OM) at magnifications up to 800X, employing both bright field (unpolarized) and polarized light. Scanning Electron Microscopy (SEM) greatly enhances the detailed observation of starch grain morphology and provides better insights into their preservation status from a structural perspective10. However, sole morphological identification is not sufficient to confirm their genuine ancient origin and remove doubts regarding the possibility of modern contamination11.Starch grains exhibit a wide range of dimensions, from a few microns to a hundred microns in width, and various shapes, with the most common ones being round, lenticular, and polygonal. They are composed of approximately 98–99% of two types of α-glucans, amylose and amylopectin, with their ratio varying depending on the plant species. These characteristics reflect their botanical origin and serve as valuable diagnostic indicators, especially for economically relevant taxa that have already been extensively studied12 and Fourier Transform InfraRed (FTIR) spectroscopy15, to demonstrate the presence of fatty acids and/or proteins. However, similar approaches have not yet been applied to identify structured U-RBRs, such as starch grains that adhere to the utilized surfaces of GSTs. Furthermore, the preservation of the chemical identity of ancient starches is also unlikely. Prolonged contact with soil constituents can lead to various biodegradation pathways, suggesting that ancient starches may possess a ‘microfossil’ nature that has yet to be revealed. A study involving Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) was conducted on modern starches to analyze the products of their non-enzymatic degradation through the Maillard reaction. This study demonstrated the potential to characterize starchy residues and their diagenetic products, and to recognize taxonomic signals, opening up new possibilities for investigating fossil contexts16. Nevertheless, the aforementioned analysis was conducted only on extracts of modern starches and not on ancient ones, which are usually much less abundant, severely limiting the application of the method.In this context, various attempts have been made to chemically identify amorphous residues on flaked stone tools using vibrational spectroscopies, such as Raman spectroscopy18. Additionally, a chemometric model was developed based on starches extracted from modern plants available in the Pontic steppe, as identified through the literary review and available pollen lists for the sites . This model was utilized for the preliminary classification of the ancient starch grains, with a majority identified as belonging to rhizomes and tubers. Overall, the methodology described in this study addresses two key aspects. First, it addresses issues regarding starch degradation and aging due to diagenetic events and their long-lasting survival. Furthermore, it demonstrates the genuine origin of starch grains obtained from GSTs dating back to at least 36,000 years ago, offering potential for their classification. From an archaeological perspective, these results are likely to demonstrate the co-occurrence of intentional processing of starchy plants with the emergence of soft technologies connected to the use of non-flaked/macrolithic tools since the early occurrence of Homo sapiens at the boreal latitudes. This contributes to our understanding of the complexity of modern human dietary habits, as their skills enabled the mechanical transformation of bio-based polymer composites, including starch, and in turn likely strengthening their capacity to overcome the constraints as a result of the harsh climatic conditions of the late MIS3 (Marine Isotope Stage).Based on the above, the innovative multi-technique approach proposed in this study aims to address this significant research gap by integrating conventional OM with SEM, Fourier Transform Infrared imaging and microscopy  to enable the identification of SU-RBRs at the individual starch grain level. Distinctive chemical features that discriminate genuine ancient starches from potential modern contaminants have been identified, primarily associated with biomineralization events. This demonstrates the long-term resistance of the structured residues and suggests possible aging pathwaysThe characterization of the limited number of starches isolated from GSTs requires for non-destructive morpho-chemical analytical approaches with single-starch sensitivity. To this aim, here we propose the exploitation of FTIR, both imaging and infrared synchrotron radiation (IRSR) microscopy, and its integration with OM, both unpolarized and polarized, and SEM for the analysis of putative ancient starches recovered from the used surfaces of nine ground stones retrieved at three sites across the Pontic Steppe: Brînzeni I (Moldova), Surein I (Crimea), and Kostenki 14-Markina Gora . More details on the geographical context of the research and the archaeological sites, in addition to the object biographies, are reported in the Supplementary Materials. Table 19. The origin of ASCs, a brief description of their isolation procedure and the analytical techniques exploited for their characterization are summarized in Table 21 and also ethnobotanical sources22, with the aim to build a chemometric model for ASCs classification.From here on, the analyzed SU-RBRs will be referred to as Ancient Starch Candidates (ASC), and ranked from ASC1 to ASC4 according to the dislodgement procedures detailed elsewhereNext, the application of the very same characterization techniques is first presented on MSR with the aim of guiding the reader on the more complex interpretation of morpho-spectroscopic results on ASCs.24.As specified in Table 8.In Fig. −1) are shown. While it is beyond the scope of this paper to detail on the spectral attribution (which may be found elsewhere26), it does seek to underpin the most relevant information useful for ASC spectroscopic analysis. On this respect, three distinctive spectral regions can be identified: i- 3600–3100 cm−1 interval, for the –OH stretching, diagnostic for water content in starches, as along with the spectral band of bending mode of water centered at about 1640 cm−1, the intensity of which increases with reduced starch crystallinity, linked also to the presence of hydroxyl groups that may undergo etherification or esterification, in time27; ii-3000–2800 cm−1 interval, for the -CH stretching of methyl and methylene groups, the spectral shape of which correlates with the relative content of the two α-glucans and the possible presence of FFAs and LPLs; iii- 1500–800 cm−1, the fingerprint region associated to the vibrations of the glucose monomers of amylose and amylopectin , the one most diagnostic for the presence of starch25. As a matter of fact, the spectra variability among the selected MSR is more evident, as well as quite pronounced, in this latter spectral range.From a chemical point of view, starch is a semicrystalline polymer, mainly composed of two polysaccharides: amylose and amylopectin. While amylose is mostly a linear chain of glucose molecules interconnected primarily by α-1,4 glycosidic linkages, amylopectin is a branched polymer with α-1,6 bridges that serve as branching points. The relative proportion among amylose and amylopectin in starches, as well as the specificities in the minimal amounts of lipids, in the form of Free Fatty Acids (FFAs) and Lysophospholipids (LPLs), as well as of proteins and minerals of which they are composed, are at the basis of the infrared classification of starches. In Fig. Figure A large number of ASC-2 were identified in the Brînzeni I samples, dislodged from two GSTs , the grinding stone was already thoroughly cleaned, hence the sonication allowed for the retrieval of few starch grains with less soil residues. Consequently, Surein I starch grains show a smoother surface when compared with the ASC-2 from Brînzeni I. SEM of the extracted Surein I starches highlights that they are mainly characterized by an oval or roundish shape and, in some cases, they show a radial fibrillary fractured surface, typical of crushed starch grains28. A peculiar conical crater was also observed in most of the Surein I starch grains.ASC-3 type starches shown in Fig. 30. The co-occurrence of starch grains together with other plant elements, e.g., phytoliths, raphides, fibers, and parenchyma, strengthens the hypothesis that these GSTs were used to process plant material, an argument already put forward by Hardy B.L. and colleagues31 following their research on the flakes from the MIS 3 sites of Starosele and Buran Kaya III in Crimea. These evidences also lessen the probability that the presence of starch grains can be ascribed as the result of modern contamination.SEM images also revealed the presence of other SU-RBR of vegetal origin diverse from starch gains Fig. m,n. Amon32.In order to go beyond the morpho-descriptive approach and to attempt the chemical characterization of the retrieved particles, the experimental plan was integrated with the label-free, non-damaging chemical characterization of the samples by using FTIRI at micrometric lateral resolution. More than 300 images were collected and processed from the ASCs extracted from the archaeological GSTs, and some of the results on select samples are shown in Fig. −1 for the OH stretching . Average spectra of ASCs were extracted from the hot-spots and some of them are presented in Fig. −1 and weak to medium –CH stretching signals, with the main peak of the methylene moieties centered between 2920 and 2925 cm−1. Both signals may be a possible marker of a different level of degradation of the polysaccharides encompassing dehydration/dehydroxylation and the loss of peripheral polysaccharide chains. For Kostenki 14 ASCs , the –OH signals above 3000 cm−1 and the –CH stretching signals in ROIs 1 and 2 respectively, are barely recognizable. The low intensity is also ascribable to the very small dimension (average diameter of a few microns) and non-aggregation of these starches, that justify the poorer spectral quality. For the latter samples, the spectral profile of ROI3 is silicate-like, while for sample 1 to 6 it is more complex and possibly deriving from the band overlap of minerals and ring vibrations characteristic of glycosidic linkage and sugar rings. In addition, FTIR micro-spectra in Fig. −1, the C=O stretching at 1790 cm−1 and the overtone at 2520 cm−137. Some clay-like signals at higher frequencies, with sharp peaks at 3612–3614 cm−1, can be seen in Fig. −1, possibly assignable to calcium oxalate crystals39. These features could not be smoothed by subtracting the contribution of the surrounding materials; therefore, it is possible to hypothesize that they are not due to soil contamination, but may be indicative of the aging associated to mineralization, partial or complete, of the ASCs. In order to prove this hypothesis, an isolation procedure of the starches compatible with FTIR analysis was developed to obtain ASC-4, as already described in19, and exploited for further and finer IRSR FTIRM analysis.ASC spectra in Fig. 41, it was possible to distinguish different morphologies and to detail diagnostic features, namely the hila and lamellae that made it possible to identify them as starch grains. The Brînzeni I starches bear a wider variety of morphologies, appreciable thanks to the larger quantity of starch grains , which include lenticular  have been identified by conventional OM inspection on the GSTs from Brînzeni I (n = 100), Surein I (n = 7) and Kostenki 14-Markina Gora (n = 4). Some optical images of recovered ASC-4 starches (both BF-OM and P-OM) are shown in Fig.  have bee−1, whereas relative intensity and positions of the bands can vary sample by sample, as already highlighted for ASC-2 and ASC-3. Isolated ASC-4 have negligible traces of soil residues and contaminants that could alter both their infrared spectrum and morphology. Therefore, since the main spectral features of ASC-4 are comparable to the ones of ASC-2 . Since P-OM and SEM undoubtedly identify these particles as starches, no doubts can be raised on their polysaccharide nature, while the mineralization of the starch, as a consequence of diagenetic processes, led to the deposition of carbonate minerals still preserving the starch original structure. In addition, the presence of sharp absorptions in the spectral range 3750–3580 cm−1, marked with an asterisk (*) in Fig. In order to characterize the vibrational profile of the isolated ASCs previously characterized by P-OM, IRSR FTIRM was carried out on the starch grains. In Fig. Any other band attribution and speculation would not be reliable and indeed not verifiable since there are no chemical complementary characterization tools that may work at the single starch level to support the band attribution. Nevertheless, ASC-2 to ASC-4 analysis allows to conclude that the spectral profile of ancient starches differs from their modern counterparts, being characterized by mineral features, indicative of the diagenetic process they underwent and support the attribution of the analyzed starch candidates as being genuine (ancient).−1, from the C–O–C asymmetric stretching, 1065 cm−1 and 985 cm−1 usually assigned to non-structural carbohydrates48. PC2 has spectral features similar to those of PC1 at low wavenumbers, with the main peaks at 1174 cm−1 from ring “breathing” vibration, 1065 cm−1 due to in-plane bending of C–OH bond and 1033 cm−1 assigned to the C–OH stretching49. At higher wavenumbers, their strong peak at 1428 cm−1 from β-glucans can be seen. PC3 presents strong signals at 1163 cm−1, 1060 cm−1 and 950, similarly to PC1 and a peak at 1740 cm−1, assigned to the C=O stretching of aldehyde group possibly from galacturonans50, and 1658 and 1622 cm−1 signals from proteins. In the high wavenumber region (3100–2800 cm−1) PC3 presents sharp peaks from CH2, probably due to the polysaccharide’s backbone or lipid aliphatic chains.In order to attempt a classification of the identified genuine ASCs, the acquired data on MSR were used to build a model suitable for categorizing the ASC, as specified in the Methods section. In Fig. −1 spectral region confirmed to be the most diagnostic for the presence of starch and, at the same time, the mostly affected by starch mineralization, the application of the identification model to the unknown ASCs performed quite well, given that all the performance indicators are higher than 0.9 (see Methods for more details) and allowed to assign to the ASCs a degree of similarity to the starch-rich organs from which they were extracted. As can be appreciated from the bar chart in Fig. Despite the 1500–800 cmWhile this analysis is not intended to be conclusive, it does prove that ancient starch identification can be attempted at the individual starch level based on an IRSR FTIRM model built on MSRs. The model must certainly be further expanded and, to increase the classification precision, it must be optimized on real train datasets of already recognized ancient starches, if any, or their closest approximation, i.e., artificially aged ones. We are working on addressing this later point in future research.To date, the direct evidence of starch-rich plant processing during the Late Pleistocene (MIS 3) has been questioned due to the inability of optical microscopy imaging techniques to confirm their genuine ancient origin. Our method, which combines imaging techniques such as optical and polarized light microscopy with scanning electron microscopy (SEM), and chemo-profiling using FTIR spectral imaging, achieves a chemical sensitivity enhanced by the superior brilliance of IRSR, allowing us to directly analyze individual starch grains.By employing it we could observe more detailed morphological features of the starch grains, such as their lamellar structure, as well as signs of mechanical processing, including, fractures, cavities, and exfoliation. Through the combination of OM, SEM, and FTIR analyses, we have achieved a groundbreaking milestone: the identification of specific chemical characteristics at the individual starch grain level, which distinguish the ancient starches extracted from ground stones dating back to MIS 3 with respect to the modern counterpart.−1 spectral region, which is also the more diagnostic of the starch botanical origin, we could classify the retrieved ASCs using a model built on MSRs as largely originating from underground storage organs (rhizomes and tubers). The chemically characterized ASC-4, which consists of isolated and purified starches from various MIS 3 sites , still display the characteristic Maltese cross. This, along with the observed mineralization features, strongly supports the distinctiveness of genuine ancient starches. These findings are in line with the data obtained from the mineralized starch grain measured in BZ#442, which can be interpreted as originating from a rhizome with an accuracy rate of at least 80% (or higher).The FTIR analysis is particularly valuable as it revealed strong bands associated with mineralized material. This enables us to differentiate between modern and ancient starches, even in complex matrices. Our approach provides evidence that the effects of aging associated with the biomineralization of starches could be observed in ASC-2 and ASC-3-type samples. Despite the mineralization features of ASCs mostly affect the 1500–800 cmIt is important to note that FTIR imaging and microscopy with IRSR operates at the individual starch grain level. This unambiguous association is a prerequisite for coupling chemo-profiling with OM and SEM. Our ability to identify single starch grains confidently helps us avoid two issues: i) confusing a genuine ancient starch with a modern one due to their different vibrational profiles, and ii) misclassifying roundish objects that show the Maltese cross as starches. Furthermore, our data has been crucial in identifying the origin of the archaeological starches, primarily as originating from underground storage organs (USOs). However, more detailed taxonomic attribution remains challenging due to the limited number of plant species available in the reference collection. Expanding the reference collection to include more wild plant species is a future goal.Overall, our research yields several significant findings. Firstly, it provides definitive answers to long-standing questions regarding the preservation and detectability of starch over millennia. Secondly, our ability to identify the genuine origin of starch found on ground stone tools from MIS 3 offers crucial evidence to support their attribution. Thirdly, our study demonstrates the feasibility of studying structured use-related biogenic residues from museum collections, providing a reliable sampling approach for future analyses. Furthermore, our study holds significance in understanding the evolutionary success of modern humans. The secure identification of genuine ancient starches, still detectable on ground stone tools, allows us to reconstruct the complex processing techniques of early modern humans exploiting diverse foodscapes. This processing included the consumption of dietary carbohydrates primarily obtained from underground storage organs (USOs). Our results strongly support the notion that starch rich food processing played a vital role in providing highly calorific nutrients during the early colonization of the West Eurasian boreal latitudes, dating back at least 40,000 years.The examined ancient starch grains were recovered from the used surfaces of nine ground stones, preliminarily identified during museum collection using low magnification stereomicroscope with magnification up to 60X. The GSTs were retrieved from three sites located across the Pontic Euxine steppe: Brînzeni I (Moldova), Surein I (Crimea), and Kostenki 14-Markina Gora (Don River), according to Table Modern Starch Reference (MSR) were prepared by grinding 5 g  of each of the selected raw storage organs by means of a blender and the chopped residue was soaked in 200 mL of ultrapure water  for 3 h. The mixture was then filtered off in a Millipore filtration apparatus (Merck Millipore Glass Vacuum filtration system) by using a metallic filter with pore size of 0.1 mm. The filtered water containing the starches was then centrifuged  at a Relative Centrifugal Force (RCF) of 6147 for 5 min at 20 °C. The pellet, obtained by removing the supernatant, was washed with 100 mL of ultrapure water and the mixture was bath sonicated for 5 min before recovering the pellet by further centrifugation (same conditions as above). The precipitated matrix was then washed with 5 mL of EtOH (Sigma Aldrich), followed by the sonication and centrifugation processes as described above and the EtOH was removed. The pellets recovered after this extraction procedure were dried and the powders obtained were stored under dark conditions.MSH Mondes in Nanterre (France) while the SEM samples were viewed at the Service de Microscopie Électronique (SME) at the Institut de Biologie Paris-Seine using a high-resolution SEM . Optical images were also collected with the Vis-IR Hyperion 3000 microscope .The archaeological samples were processed in two different institutions (CNRS in Nanterre (France) and IHAE FEB RAS in Vladivostok, Russia) and observed with different optical microscopes with both unpolarized and polarized light . The MSR were collected, prepared and extracted at DAIS in Venice and the OM images were visualized at the ArchéoScopie Platform of the Optical Microscopy, OM).In accordance with Table Droplets of 0.05 mL were deposited on ZnSe windows, left to dry, and scanned uncoated to guarantee the reliability of the complementary analytical steps, like FTIR and possibly other analytical techniques .51. FTIRI with transmission sampling geometry was applied on ASC-2 and ASC-3 samples: the microscope is equipped with a 64 × 64 pixel Focal Plane Array (FPA) detector capable of acquiring a full FTIR spectrum per pixel, thus generating 4096 pixels’ hyperspectral images for each measure. Given the 15× magnification of the used Cassegrain objective, the pixel size is about 2.6 × 2.6 microns and the field of view of one image tile is about 167 × 167 microns. Mosaics containing multiple tiles were also acquired for each sample. The instrumental parameters used for FTIRI measurements were the following: 64 scans at 8 cm−1 spectral resolution, 5 kHz scanner speed. More than 300 hyperspectral maps were collected. Isolated ASC-4 starches were measured with the same instrument by FTIRM in transmission sampling geometry, using Infrared Synchrotron Radiation (IRSR) source and a Mercury Cadmium Telluride (MCT-A) detector. The parameters used for each IRSR measurement were the following: 512 scans at 4 cm−1 spectral resolution, 120 kHz scanner speed, setting the apertures at the same size of the starches, from 20 × 20 to 40 × 40 microns. For both FTIRI and IRSR FTIRM measurements, the background was collected on a clean portion of the window support, with the same parameters used for the sample data collection. For MSR, the spectra have been acquired with FTIRM with conventional source, averaging 128 scans for each spectrum at a spectral resolution of 4 cm−1 setting the scanner speed at 40 kHz. With the MSR being much more abundant than ASCs and being deposited in a densely packed layer, the lateral resolution was set at 50 × 50 microns.ASC-2 to ASC-4 and MSR samples (as detailed in Table https://quasar.codes)53. FTIRI and FTIRM data were pre-processed by applying the water–vapor compensation routine of the proprietary Bruker software OPUS 8.5. Due to spectral interference from surrounding material, of mineral origin mainly from the soil, ASC-2 spectra had to be further pre-processed before interpretation by subtracting this contribution: the spectrum of the surrounding material was obtained by extracting an average spectrum from pixels adjacent to the target particle. More details on the method are reported in the caption of Supplementary Fig. Both FTIRI and FTIRM data were analyzed in OPUS 8.5 (Bruker Optics) and Quasar (https://quasar.codes). MSR were preprocessed as follows: interpolation to a common spectral range and data spacing, baseline correction with a rubber band method, derivatization using the Savitzky-Golay algorithm set at 23 window points 3rd degree polynomial and 2nd grade derivative, and vector normalization. This data was then reduced using a principal component analysis (PCA) to 16 principal components that represent the 95% of the total variance. Within this 16-dimensional PCA space, k-Nearest Neighbor (kNN) algorithm was used with 4 points and the weighting distance calculated as Euclidean distance. The performances of the obtained model were evaluated by stratified cross validation repeated 6 times. The performance indicators are the following:Area under the Curve (AUC): 0.990.Classification Accuracy (CA): 0.925.F-1 Parameter: 0.922.Precision: 0.927.Recall: 0.925.A chemometric model was built using MSR spectra as training dataset. The model was developed in Quasar (Supplementary Information.


As new sources of proteins, edible insects may be excellent additives in a new generation of environmentally friendly food products that are nutritionally valuable, safe, sustainable, and are needed in today’s world. The aim of this study was to determine the effect of the application of cricket flour on extruded wheat-corn-based snack pellets’ basic composition, fatty acids profile, nutritional value, antioxidant activity and selected physicochemical properties. Results showed that the application of cricket flour had a significant impact on the composition and properties of snack pellets based on wheat-corn blends. In newly developed products, the enhanced level of protein and almost triple increase in crude fiber was found as an insect flour supplementation reached 30% level in the recipe. The level of cricket flour and the applied processing conditions (various moisture contents and screw speeds) significantly affect the water absorption and water solubility index and texture and color profile. Results revealed that cricket flour application significantly increased the total polyphenols content in the assessed samples in comparison to plain wheat-corn bases. Antioxidant activity was also noted to be elevated with increasing cricket flour content. These new types of snack pellets with cricket flour addition may be interesting products with high nutritional value and pro-health properties. Food and Agriculture Organization of the United Nations promote edible crickets as a way to alleviate the food crisis as more sustainable and environmentally friendly than farming animals, such as chickens, pigs, and cattle ,2,3. It Acheta domesticus L., is important industrialized insect species in the world  and mg—gel mass [g].WAI was determined as described by Wójtowicz and Mościcki . The grodv—vessel mass after drying [g], mv—vessel mass before drying [g], and ms—dry sample mass [g].Water Solubility Index was determined as described by Wójtowicz and Mościcki . The liq−1. The cutting force was registered as the highest force (N) when the pellet became completely broken. The tests were performed in five replications.The cutting force of snack pellets was tested with Zwick/Roell apparatus BDO-FB0.5TH  based on Jin et al.  methodolΔE) using the NR20XE colorimeter . Ground samples of snack pellets were tested for lightness L* ranging from 0 (black) to +100 (white), redness a*, and yellowness b* in five replications each [The profile of color coordinates in the CIE-Lab scale was evaluated and the total color change index  was used to analyze the results. The obtained data were analyzed with one-way (ANOVA) analysis of variance followed by the post hoc test with the Fisher’s LSD (least significant difference) at the significance level of α = 0.05 to assimilate mean values.Pasting characteristics may be useful for the indirect estimation of the extrusion-cooking treatment intensity of raw materials with significant content of starch. Peak viscosity indicates the ability of raw materials composition to water absorption and starch swelling and is influenced mostly by starch content in blends. Based on the results of the pasting profile of tested compositions , increasHot paste viscosity (HPV) indicates the sensitivity of the swollen starch granules to shearing and disintegration. Mitrus et al.  reportedFirst, the proximate composition and fatty acids profile of the selected extrudates were determined . Both thApplication of 30% of cricket flour to the extruded recipes increased the content of basic chemical components, such as crude protein, crude ash, crude fat, and crude fiber, in comparison to control samples and products containing 10% of insect flour, for both screw speeds (60 and 100 rpm) applied during processing. The high content of total protein–a valuable dietary ingredient for humans, in insects, has been described in many studies ,41,42,43Acheta domesticus) was included in extruded snacks, while García-Segovia et al. [In the control sample, the protein content reached 9.18–9.33 g/100 g, ash content was 2.85–2.91 g/100 g, ether extract was 0.33–0.80 g/100 g, fiber was 0.00 g/100 g, and carbohydrate content was 76.99–77.39 g/100 g. The highest levels of proximate components were found in snack pellets supplemented with 30% of insect flour additions. Here, protein content reached 24.07–24.60 g/100 g, fat 0.12–0.36 g/100 g, ash 4.19–5.64 g/100 g, crude fiber 5.92–7.22 g/100 g, and carbohydrates at 71.02–75.55 g/100 g . Ramíreza et al.  found inOur research showed that increasing the content of cricket flour as a component in the production of extruded snack pellets from 10 to 30% did not significantly affect the PUFA content while increasing the SFA content in the total fatty acids profile . This is comparable to the outcome of the work of Raksakantong et al. , who notThe lower-than-expected crude fiber content in extruded snack pellets could be caused by the process of degradation of macromolecular compounds as a result of the extrusion-cooking process and the related impact of high temperature and pressure on the raw material ,49. StilBased on the performed study, insect flour can be inferred as a component with great potential to influence the nutritional value of snack foods due to its high protein and fiber content in snack pellets.Total phenolics content (TPC), as assessed via the Folin–Ciocalteu method, is a helpful parameter for determining the antioxidant potential of plants or foodstuffs extracts. The assay is used to estimate the amount of contained high active antioxidants . These authors reported moderate scavenging action depending on the extraction solvent, with the lowest IC50 (0.119 mg/mL) for the methanolic extract. In contrast, aqueous extracts of H. parallela revealed significant antioxidant activity (IC50 of 1.45 mg/mL) [T. molitor and A. domesticus extracts, which showed a clear relationship between a higher content of polyphenols and a higher scavenging capacity of DPPH [Tenebrio molitor) and cricket (Gryllodes sigillatus) flour based on the determination of total phenolic content (TPC) and the ability to neutralize DPPH· and ABTS· free radicals. They also found that this fortification resulted in a lower predicted glycemic index, which makes the addition of insect flour favorable in products for people who often struggle with various conditions, such as obesity, diabetes, and insulin resistance [In the course of our research, the antioxidant potential of cricket flour-enriched snack pellets extracts were evaluated via DPPH radical assay. The outcome confirmed the promising antioxidant capacity of the product. Our results can be compared with the results presented by Suh et al. , based o5 mg/mL) . In othe of DPPH . In the  of DPPH  when tessistance . Previously, the antioxidant activity of insects was associated only with the presence of polyphenols. However, bioactive peptides, proteins, and free amino acids coming from insects may also redound to the antioxidant activity of the tested extracts ,55,58. FThe increase in the antioxidant capacity of snack pellets may come from the presence of the active compounds in them and from the method of cricket farming and cricket product preparation. According to Zielińska et al. , the theTherefore, an important part of further research will be to confirm the absorption of these secondary plant metabolites from food, and the effect of these compounds on the composition of products enriched with insects. Studies on the bioavailability of compounds can significantly increase interest in entomophagy. The new knowledge will be an added value in favor of consuming insects or insect-enriched foods. The results of the Water Absorption Index (WAI) and Water Solubility Index (WSI) of snack pellets enriched with cricket flour are presented in In snack pellets samples, with the addition of cricket flour, a lowering of WAI was noted. This is probably related to the increased protein content and, thus, decreased the total carbohydrates content (from 76.99–77.39% in the control sample to 52.16–52.40% when the insect flour content was 30% in the recipe. A lowered content of carbohydrates, especially starch, is responsible for lowered WAI, and this has a connection to low starch gelatinization. Application of cricket flour at the level of 10 and 30% showed a significant decrease in WAI, especially when low moisture was used, and low screw speed was applied during snack pellets extrusion. We observed that while screw speed had an effect, strong dependencies were also observed when varied initial moisture levels were employed. Here, increased water level availability for transforming and gelatinizing the starch in all tested samples increased water absorption independently of screw speed and insect flour content. This means that the water added to raw materials had a crucial role in starch gelatinization and, thus, in WAI in the tested samples. A low WAI level in the snack pellets indicates the degree of starch gelatinization and can affect the expansion procedure, especially without frying. This effect may be notable, especially in the microwave or hot air processing . The WAILowering the starch content in the recipe by the application of cricket flour also affected the WSI results. WSI allows us to measure soluble components not compacted within the starch-protein matrix formed during the extrusion-cooking and results mostly from starch degradation but also from the presence of non-soluble components in the recipe. The results of WSI obtained for the snack pellets of our experiment showed similar tendencies when no and 10% of cricket flour was added into the wheat–corn-based recipe . Higher The texture of snack pellets evaluated as the cutting force is presented in L* values were observed with any significant differences in lightness due to processing conditions. The changes in the snack pellets’ color mainly resulted from the increase in the content of cricket flour characterizing brown color coming from insect carapace [a* was noted in control samples processed at 60 rpm. Slight changes were observed in the red tint of control snack pellets processed at 100 rpm and when 10 and 30% of cricket flour were applied, not dependently on the processing conditions. Snack pellets containing 30% of cricket flour were less intensively yellow than those supplemented with 10% of edible insects, and double lower yellowness intensity was noted as compared to control samples. b* values were found from 24.38 for the control sample, which was yellow due to the presence of corn flour up to 12.45 at the highest level of cricket addition replacing yellow components and becoming browner then yellow. In this case, slightly more intensive yellow tint was observed in samples processed at higher screw speeds and higher initial moisture content. All these color indicators had great influence on the total color change index ΔE, when it is above 3, it means that color can be easily differentiated by eyes from the reference sample [The results of color coordinate and the total color change are presented in carapace . The lowe sample .ΔE ranging from 13.10 to 21.88. Much lower changes in color were found by Lisiecka et al. [ΔE was max 2.16 in pellets and 7.97 after microwave expansion. However, the total color change may be an effect of other processes occurring during the extrusion-cooking, such as Maillard reactions, caramelization, or degradation of pigments [Application of both levels of cricket flour significantly changed the total color of snack pellets, with values of a et al.  testing pigments .The cricket flour addition in wheat-corn blends had a significant impact on the proximate composition of snack pellets processed with the extrusion-cooking. Enhanced levels of protein from 9.3 to 24.6 g/100 g were found if cricket flour content was boosted from 10 to 30% of the total components in the recipe. Indeed, an almost triple increase in crude fiber was noted if insect flour was used at a 30% level. Processing conditions showed a slight effect on the chemical composition of extruded snack pellets. Cricket flour content at either 10 or 30% of total flour content had a significant effect on snack pellets WAI and WSI. Here, the WAI decreased at low initial moisture and low screw speed during snack pellets extrusion. Cricket flour addition lowered snack pellets’ WAI and lightness, while WSI increased with increasing initial moisture and with higher screw speeds and the highest cricket flour addition. This indicates that components were not sufficiently integrated into the snack pellets structure. A similar conclusion can be stated based on cutting force results, which were significantly lower with an increased amount of cricket flour in the recipe. Such an outcome was probably due to high crude protein and crude fiber content and, thus, low starch content. Moreover, results showed that cricket flour addition significantly increased total polyphenols content in comparison to the wheat–corn control sample. The antioxidant activity of snack pellet extracts increased with increasing cricket flour content in the recipe. Therefore, it can be concluded that cricket flour shows great potential to enhance the nutritional value of newly developed food products due to the high protein and fiber content in snack pellets.


Arterial stiffness is a disease of the arterial media, and it is well known that it is accelerated by aging. Arterial stiffness, vascular biomarkers such as central hemodynamics, wave reflections, and endothelial function, is associated with cardiovascular diseases (CVD) such as hypertension, diabetes, and dyslipidemia due to changes in the mechanical properties of the arterial walls. A recent epidemiological study  investigRecently, based on this concept, Anastasio et al. , in the It has been established that arterial stiffness is determined by chronic physiological factors: age; blood pressure; and unhealthy lifestyles such as an inappropriate diet, a lack of physical activity, and smoking-induced reduction in the elasticity of the arteries due to a structural change of the arterial wall . HoweverChronic exercise is effective in reducing arterial stiffness in the long term. On the other hand, an acute effect of exercise on the arterial stiffness index has been observed, although it returned to the baseline level in the following 24 h . This fiWith this background, we need to point out two matters. First, the measurement of arterial stiffness should be an important indicator of the index of risk of cardiovascular disease (CVD); however, the difference between acute and chronic effects or between functional and structural effects should be distinguished in order to assess arterial stiffness. In the other words, it may be considered that chronic effects on arterial stiffness need to be isolated from acute effects if CVD risk is evaluated. Second, many researchers have investigated the effect of acute interventions, such as exercise, on arterial stiffness based on the concept that a repeated acute effect causes a chronic effect. However, the acute effect on arterial stiffness is completely different from the chronic effect; the acute effect may be caused by functional modification, but in contrast, the chronic effect may be caused by a structural effect. Thus, it remains unclear whether repeated acute changes in arterial stiffness have chronic effects.


The objective of this study was to investigate the effect of temperature changes during heat–moisture treatment (HMT) on the appearance, structure and digestibility of sweet potato starch (SPS). The results showed that after HMT, there were depressions, cavities and fragments on the surface of SPS particles. The polarized crosses of SPS were irregular and partially blurred. The relative crystallinity and short-range order of SPS decreased, while rearrangement and reorientation of the starch molecules occurred and the thermal stability increased. The resistant starch content of SPS reached the highest (24.77%) after 4 h treatment at 110 °C and 25% moisture. The obtained results can provide a reference for the modification of SPS. People’s poor eating habits have led to the incidence rates of chronic diseases rising year on year, tending to affect younger individuals ,2. The uIpomoea batatas) is an annual or perennial herb of Convolvulaceae [Sweet potato  technology was first proposed by Sair in 1967 . It has At present, studies on HMT of starch are mainly focused on rice starch and wheat starch ,15. TherSPS was purchased from Haihua Shunda Grain and Oil Seasoning Co., Ltd. . Its protein, fat, moisture, mineral and starch contents were 0.22 ± 0.03%, 0.16 ± 0.01%, 12.77 ± 0.26%, 0.11 ± 0.01% and 84.52 ± 0.17%, respectively. Its amylose content was 27.73 ± 0.85%. α-Amylase from porcine pancreas (50 U/mg) and glucoamylase (80 U/mg) were obtained from Bomei Biotechnology Co., Ltd. . Other chemicals  were of analytical grade.According to the method of Zhang et al.  with sliAccording to the method of Jhan et al.  with sliA 0.65% starch solution was dripped on the slide, covered with glass, and placed on the carrier platform. The polarization properties of starch were observed by a BH200P polarizing microscope  under polarized light amplification (200 times).An appropriate amount of the SPS sample with pre-balanced moisture was tiled in the sample pool, and the excess sample was scraped to ensure a crack-free sample surface and placed on the XRD sample table. The crystal structure of starch was determined by a D8 Advance X-ray diffractometer, . The XRD pattern of the sample in the 2θ range of 4~40° was recorded with a scanning rate of 2°/min at 40 kV and 40 mA. The crystallinity of the samples was calculated by MDI jade 6.0 software .−1 was obtained after scanning 32 times with KBr as the background. The IR spectrum was convoluted by OMNIC software . The spectrum (1200–800 cm−1) was selected with an enhancement factor of 1.9 and a half-peak width of 19 cm−1. Peakfit 4.12 was used for peak separation. Then, the intensity ratio of the peaks at 1047 cm−1 and 1045 cm−1 was calculated.The short-range ordered structure of the sample was evaluated based on the method of Wang et al. ; 1 mg of0), peak temperature (Tp) and end temperature (Tc) of the test samples were recorded.The thermal parameters of the sample were measured by a Q2000 differential scanning calorimeter  . The samG20 and G120 are the glucose contents after enzymatic hydrolysis of 20 min and 120 min, respectively; FG is the glucose content in the sample before enzymatic hydrolysis; and TS is the total starch content in the sample.The in vitro digestibility of the SPS sample before and after HMT was determined according to the method of Englyst . The drip < 0.05).All tests were repeated 3 times and results were expressed as means ± standard deviation (SD). SPSS 26.0 software was used for single factor variance statistical analysis, and Origin 2017 software was used for plotting. The statistical comparison was based on the Tukey method with a confidence level of 95% , under the action of heat energy and water molecules, the amylopectin molecules inside the starch molecules degraded. As a result, the starch chains recombined to form an ordered structure  with a higher thermal stability, which enhanced the rigidity of the starch structure, resulting in an increase in the pasting temperature of starch [The DSC curve reflects the change in thermal stability of starch particles during heating. As shown in f starch . HoweverThe different digestion rates of starch in the human body are generally divided into RDS, SDS and RS . Table 3This study investigated the effect of temperature changes during HMT on the structure and in vitro digestibility of SPS. The results indicated that temperature was one of the most important factors affecting the modification of SPS, having a significant impact on the structure of SPS. After HMT, the surface of SPS granules was destroyed, resulting in fracture, depression and adhesion. Although the polarized cross of SPS was retained, the arrangement was irregular. HMT did not change the original crystallization type of sweet potato starch (A-type), but the diffraction peak at 19.8° was more pronounced. The relative crystallinity and short-range order of SPS decreased gradually, but the thermal stability increased. The in vitro digestibility of SPS changed with the increase in HMT temperature in an overall downward trend. The RS content reached its highest (24.77%) when the temperature was 110 °C at 25% moisture for 4 h. This was also the most ideal temperature for HMT of SPS. This study provides a theoretical basis for the preparation of RS using HMT and also provides a reference for the development of SPS products.


Triticum turgidum subsp. durum) is widely grown for pasta production, and more recently, is gaining additional interest due to its resilience to warm, dry climates and its use as an experimental model for wheat research. Like in bread wheat, the starch and protein accumulated in the endosperm during grain development are the primary contributors to the calorific value of durum grains.Durum wheat ), then RNA sequencing was used to profile the transcriptome at each stage. The largest changes in gene expression profile were observed between the earlier timepoints, prior to 15 dpa. We detected a total of 29,925 genes that were significantly differentially expressed between at least two timepoints, and clustering analysis revealed nine distinct expression patterns. We demonstrate the potential of our dataset to provide new insights into key processes that occur during endosperm development, using starch metabolism as an example.We provide a valuable resource for studying endosperm development in this increasingly important crop species.The online version contains supplementary material available at 10.1186/s12870-023-04369-7. The endosperm constitutes about 80% of a wheat grain by weight, and is rich in storage reserves that are mobilised during seed germination to nourish the developing embryo . Starch The endosperm originates from double fertilisation, where a pollen nucleus fuses with the two polar nuclei of the central cell , 4. The Starch is synthesised in amyloplasts of starchy endosperm cells from sucrose imported into the developing grain from source tissue. It exists as semi-crystalline, insoluble starch granules that are composed of two distinct glucose polymers - amylopectin and amylose. However, wheat (and other species of the Triticeae) is unique among cereals in that it has a “bimodal” distribution of starch granules, where two types of starch granules arise from a distinct temporal pattern of granule initiation . A-type Triticum aestivum), relatively fewer resources exist for tetraploid durum wheat (Triticum turgidum ssp. durum). Durum wheat is widely grown in semi-arid climates, primarily for pasta production. Worldwide, it represents ≈5% of wheat grown, but it is still the 10th most widely cultivated cereal crop [in silico TILLING mutant collection in variety Kronos allows easy isolation of mutants for functional genomics, and mutations in only two homeologs need to be combined for most genes [While many studies have focused on grain development in hexaploid bread wheat , we generated a transcriptomics dataset from endosperm tissue extracted throughout grain development, in variety Kronos. Developing grains were harvested from plants grown in controlled environment chambers - starting from the earliest timepoint in which the starchy endosperm could be reliably dissected from the pericarp (6 dpa) and until the grain turned yellow, indicating maturation (30 dpa)Fig. A.We measured the starch content of these grains throughout endosperm development, which follows a sigmoidal pattern Fig. B. A logikallisto [RNA was extracted from endosperm tissue dissected from the grains (n = 3), with each replicate using multiple grains (3–6 depending on the timepoint) harvested from an independent plant. RNA sequencing (RNA-Seq) using Illumina technology yielded between 78,762,223 and 169,888,168 reads per sample. These data were pseudoaligned using kallisto . A rangeWe also calculated TPM values for all annotated transcripts in the Svevo v1 transcriptome, rather than using the Ensembl canonical transcripts only, as a resource for future analysis  to visualise and identify samples that had similar gene expression patterns Fig. C. Replic2fold change > 1.5) for all pairwise comparisons of the sampled timepoints  will be referred to as the ‘global clusters’ hereafter. Several global clusters represented general up- or down-regulation over endosperm development . However, the remaining global clusters were more dynamic and may be associated with processes that act during a specific period of development. Most of the genes were grouped into cluster 0 (5477 genes), which follows an increasing pattern similar to the starch accumulation curve  genes (DEGs) were calculated using DESeq2  metabolism, plastidial sugar transporters and starch granule initiation  , 26. TheWe also observed that durum wheat has only one set of homeologous phosphoglucomutase (PGM) genes, similar to what was shown for hexaploid wheat . Both hoAmylopectin is the primary constituent of starch and is a highly branched polymer with ⍺-1,4-linked glucose chains and frequent ⍺-1,6-linked branches. Its synthesis requires coordinated action of Starch Synthases (SS) that elongate the polymer chains, Starch Branching Enzymes (SBE) that introduce the branch points, and Debranching enzymes (primarily Isoamylases - ISA) that ensure correct branch pattern formation . Most DEHomeologs of Early Starvation1 (ESV1) and Like Early Starvation (LESV), ESV1-B and LESV-A, were unique within this category in that they clustered in C2, peaking in expression during mid-grain development (13–15 dpa). ESV1 and LESV were discovered in Arabidopsis and are hypothesised to organise amylopectin within the starch granule . Their eptst1 mutants produce amylose-free starch [Amylose is the minor polymer of starch and consists of α-1,4-linked linear chains with very few branches, and is synthesised by a specialised SS isoform, the Granule Bound Starch Synthase (GBSS) . Wheat se starch . Howevere starch . The lowCereal starch is almost devoid of covalently bound phosphate . Consistphs1 mutations can affect starch granule numbers per chloroplast if other components of MOS metabolism are simultaneously abolished [phs1 knockout mutants are severely shrivelled, particularly when grown at low temperature [Two plastidial enzymes involved in processing MOS were expressed in the endosperm: ⍺-glucan phosphorylase (PHS1) and disproportioning enzyme (DPE1) - with PHS1 peaking at around 10 dpa (and appearing in C1) while DPE1 peaked later . PHS1 activity has been observed in the developing wheat endosperm, where it was proposed to participate in starch synthesis either by trimming glucan chains at the granule surface, or by degrading MOS released by ISA during starch synthesis . It is abolished . In riceperature . The enzperature . Since tperature .By contrast to the plastidial PHS1 and DPE1, their cytosolic counterparts, DPE2 and PHS2, showed very low expression. It is plausible that the activity of these enzymes is not important in the developing endosperm, given that cytosolic MOS processing is typically associated with the further metabolism of maltose generated during starch degradation in leaves , 56.We observed very low expression of the plastidial maltose exporter, MEX1, consistent with there being low starch degradation to generate maltose for cytosolic processing. Interestingly, we observed substantial expression of the glucose transporter pGlcT. In Arabidopsis leaves, pGlcT works together with MEX1 to export products of starch degradation . Its rolThe low expression of most starch-degrading enzymes is consistent with the endosperm being primarily a site of starch synthesis. The major plastidial α-amylase and isoamylase activities, AMY3 and ISA3, were filtered out due to very low expression at all timepoints  may serve indirect functions in starch synthesis, for example by supplying MOS substrates to starch biosynthesis enzymes (see MOS metabolism above). One hypothesis is that they could release glucans from A-type granules, providing substrates for the initiation of B-type granules.A distinct set of proteins involved in starch granule initiation in Arabidopsis has recently been discovered. However, research investigating their roles in mediating the distinct spatiotemporal pattern of A- and B-type granule initiation in wheat is still on-going , 67. TheSo far, the Mar1 Filament-like Protein (MFP1) is the only known granule initiation protein identified in Arabidopsis that has not been explored in wheat. Interestingly, all cereals have two paralogs of MFP1 (MFP1.1 and MFP1.2) , which w(Triticum aestivum) [TaBGC1 compared to the other granule initiation genes  , we consnes Fig. B. HoweveTo assess how specific these expression patterns are to species that have A- and B-type granules, we examined the expression patterns of these genes in the endosperm of rice and maize using publicly available data. Rice produces compound granules, where granules are initiated during early grain development and there is no second wave of granule initiation in later grain development that leads to B-type granules; and maize produces simple granules without distinct populations of different granule sizes . Among tWe have generated a high-quality transcriptomics dataset for endosperm development in durum wheat, and we demonstrated its potential to provide new insights and hypotheses into key processes during grain development, using starch metabolism as an example. Other processes to be investigated may include protein and lipid biosynthesis, cell wall biosynthesis, micronutrient transport, as well as development of endosperm structure and subsequent maturation in general.− 2 s− 1. Each spike was individually marked for anthesis when an anther was visible from the spike, and this was taken as day 0. Checking for anthesis and harvesting were performed at 1–2 h after midday. Four spikes from each individual plant were used, each for a different developmental time point. Three biological replicates per time point were collected from independent plants. Therefore, some of the biological replicates for separate time points were spread over several of the same plants. We ensured that replicates for every time point were spread across different spikes in each plant, so that variability between spikes would not be biased towards one time point. Although there were slight differences in the day of flowering between spikes, many plants had spikes that flowered within a few days of each other and in our optimal growth conditions, the first 3–4 tillers look similar. Any variability between tillers and spikes is not likely be a huge variable for our experiment, as the quality control steps demonstrated that the principal difference separating the samples was their developmental stage  at 60% relative humidity with 16 h light at 20 °C and 8 h dark at 16 °C. The CER light intensity was 400 µmol photons mTotal RNA was extracted (n = 3) from the frozen endosperm tissue using the RNeasy PowerPlant kit (Qiagen). An on-column DNase digest was incorporated during the extraction. Either 6 , 4  or 3 (for 20 and 30 dpa) endosperms were pooled from each spike for each RNA extraction. Quality control, poly-A selection library preparation and RNA sequencing (RNA-Seq) using a NovaSeq 6000 machine (Illumina) was carried out at Novogene . Each sample returned between 78,762,223 and 169,888,168 million clean 150 bp paired end reads. These clean RNA-Seq reads can be downloaded from the NCBI Gene Expression Omnibus repository (Accession: GSE216253).kallisto [DESeq2 [DEapp shiny [2 fold change > 1.5 and a minimum count cut-off of 10 counts per million in at least three samples).Clean reads from above were pseudoaligned to the Svevo v1 Durum wheat transcriptome downloaded from Ensembl Plants, either using canonical transcripts only  [Clustering analyses were performed using Clust , on all isation) . Paramet2 TPM values for the three replicates at each time point were used as the input for heatmap and hierarchal clustering analyses. Analyses were carried out in version 1.0.12 of the pheatmap package, running on R version 4 (https://cran.rstudio.com/web/packages/pheatmap/index.html).Principal component analysis (PCA) was carried out in R first using the ‘prcomp’ function from the ‘stats’ package to generate the components of the PCA, then plotted using the ‘ggbiplot’ function from the ‘ggbiplot’ package. TPM values were used as the input for the PCA, with genes with a TPM over one for all replicates being included. For heatmap visualisation and hierarchal clustering, the R function ‘pheatmap’ from the ‘pheatmap’ package was used. Only the rows (genes) were clustered and not the columns (timepoints), otherwise default settings were used. Average logGenes related to starch synthesis in wheat were manually curated: Orthologs in durum wheat were identified using BLASTp against the Svevo v1 Durum wheat proteome on Ensembl Plants , as wellFor qRT-PCR, we generated cDNA using the same RNA samples described above for RNA-seq, using the Go-Script Reverse Transcription System (Promega). The amount of RNA in each sample was measured using a Nanodrop, then 500 ng of each sample was used for cDNA synthesis.Five starch metabolism genes  were selected for a qRT-PCR assay to check whether this was consistent with our RNAseq expression patterns. We chose four genes which were also selected in the hexaploid wheat RNAseq study : AGPS1, We performed qRT-PCR using the SYBR Green Jumpstart Taq ReadyMix (Sigma-Aldrich), using 12 µL reactions. First, we performed primer efficiency tests using cDNA from two time points, 13 dpa and 18 dpa. This was done using 5 dilutions of the cDNA samples. Using the 13 dpa sample, the primer efficiencies were all between 95 and 105%, except for the BGC1-A1 primers  The parameters of the model were extracted with the coef function.For curve fitting to the data, the nls function with the SSlogis model in R (version 4.2.0) was used. This was plotted with the ‘growthcurve’ package


The aim of this work was to assess the association between graft-to-recipient weight ratio (GRWR) in adult-to-adult living donor liver transplantation (LDLT) and hepatocellular carcinoma (HCC) recurrence. A search of the MEDLINE and EMBASE databases was performed until December 2022 for studies comparing different GRWRs in the prognosis of HCC recipients in LDLT. Data were pooled to evaluate 1- and 3-year survival rates. We identified three studies, including a total of 782 patients (168 GRWR < 0.8 vs. 614 GRWR ≥ 0.8%). The pooled overall survival was 85% and 77% at one year and 90% and 83% at three years for GRWR < 0.8 and GRWR ≥ 0.8, respectively. The largest series found that, in patients within Milan criteria, the GRWR was not associated with lower oncological outcomes. However, patients with HCC outside the Milan criteria with a GRWR < 0.8% had lower survival and higher tumor recurrence rates. The GRWR < 0.8% appears to be associated with lower survival rates in HCC recipients, particularly for candidates with tumors outside established HCC criteria. Although the data are scarce, the results of this study suggest that considering the individual GRWR not only as risk factor for small-for-size-syndrome but also as contributor to HCC recurrence in patients undergoing LDLT would be beneficial. Novel perfusion technologies and pharmacological interventions may contribute to improving outcomes. Hepatocellular carcinoma (HCC) is the most common primary liver cancer with increasing incidence and estimated death rate of 55% until 2040 ,2. SinceSome early reports demonstrated advantages for LDLT over deceased donation, when considering patients with HCC, findings that were further supported by several prospective intention-to-treat analyses, where the overall recipient survival was comparable between living and deceased liver recipients ,5. A fewAnother inherent advantage appears with the shorter waiting times for candidates where a living donor is available. Goldaracena et al. demonstrated that patients who had a potential live donor at the time of listing had a higher survival rate . InteresDespite such results and the general opinion that LDLT imposes a lower risk and better outcomes in liver recipients, some other authors have also reported controversial data. Fisher et al. presented inferior oncological outcomes after LDLT compared to deceased donor transplantation, as patients undergoing LDLT had higher HCC recurrence rates within 3 years . Such fiThe overall controversial clinical results also point to confounders, other than recipient cancer risk, that may have impact on recurrence rates. Donor risk factors , surgical parameters and stress  and the recipient risk  are just a few adjunct variables, which add to the already evident recurrence risk conveyed by the candidate’s liver cancer status and biology ,13. SuchDespite the generally better quality of living donor liver grafts, compared to the deceased donor pool, the overall strategy is to protect the donors and to avoid the development of small-for-size-syndrome (SFSS). Per definition the development of a SFSS represents a situation where a small graft shows a primary dysfunction within the first postoperative week after transplantation without any sign of other pathologies  . While tAdditional risk factors may contribute to the observed SFSS, including the transplanted liver graft volume and the cytokine release triggered by both liver transection during donation surgery and after reperfusion . The mecBased on the above-mentioned concerns, the aim of this study was to perform a systematic literature review of all studies that reported data on oncological outcomes after LDLT for HCC and provide details on the association between liver graft rate recipient weight ratio (GRWR) and oncological outcomes.A total of 45 papers were identified and screened. In 16 studies, the vast majority of authors reported their survival and SFSS-rates after LDLT using a cut-off for GRWR at <0.8% vs. ≥0.8%  19,20,2,32,33,34The included studies reported a total of 782 patients; 168 underwent LDLT with a GRWR of <0.8%. Conversely, 614 were recipients, who received a liver with a GRWR of ≥0.8%. All patients were diagnosed with an HCC.The mean donor age ranged between 26 and 33 years. The recipient age was found to be between 47 and 54 years, with the majority being male (range 76–98%), in the overall cohort. The baseline characteristics are summarized in From this pool of literature, three studies met the inclusion criteria . Such stThree studies reported the graft types ,36,37. ILee et al.  exclusivOne study  reportedThe risk of bias assessment of the included studies is demonstrated in One study  reportedOne study reported  the 1-, This systematic review focuses on patients undergoing living donor liver transplantation (LDLT) for hepatocellular carcinoma (HCC). The overall number of suitable studies that transparently report the graft volume together with the candidate’s HCC cancer status with enough follow-up is limited to three. LDLT using small grafts with a graft recipient weight ratio (GRWR) of <0.8% was associated with lower overall and particularly tumor-free survival rates in patients with higher tumor burden outside Milan criteria. Although there were only three studies with a relatively small number of cases, this systematic review points to an outcome benefit considering donor liver volume in HCC recipients, particularly in candidates outside standard tumor criteria. LDLT has emerged as standard treatment, especially in most Asian countries where the deceased donor transplant program is limited or not active . LDLT ofAvailable intention-to-treat analyses have demonstrated a survival advantage for living donor liver recipients over DDLT ,8,42,43.Although a GRWR of >0.8% is preferable, levels GRWR between of ≥0.6% and 0.8% can be acceptable if an alternative living donor is lacking, for primary transplants of recipients with maximal one organ failure. For this approach, both the donor and recipient should consent to accept the higher risk  and an iThe relevance of blood flow and shear stress is well-described as trigger of endothelial cell dysfunction in different vessels. Disturbed blood flows with subsequent reduced shear stress at the endothelial cells, as seen with relevant portal hypertension in small liver grafts, may significantly impact mitochondrial function and increase mitochondrial ROS release with inflammation, thereby blocking mitochondria from their essential task to produce ATP—the key molecule needed for the full metabolic function of a liver, which can quickly regenerate without severe SFSS .Based on the increasing surgical experience during the past few decades, various surgical techniques to modulate the liver inflow now exist, including splenic artery ligation, splenectomy or portocaval shunts. Such surgical steps are often required when the GRWR is below 0.7%. Intraoperative decision making is based on the portal vein and hepatic artery flow and pressure measurements . Other fThe Cleveland group has recently demonstrated that an augmented graft outflow reconstruction  together with meticulous PVP/flow measurement and inflow modulation whenever appropriate (before or after graft implantation) can lead to excellent and comparable results after LDLT using small grafts (GRWR < 0.7) .Although living donor grafts are carefully selected and of optimal tissue quality, cells are subjected to a few more minutes warm ischemia at the time of recovery. Considering that such grafts undergo several blood-inflow clamping cycles (Pringle maneuver) during the transection of the donor liver, which activates the IRI-cascade, there is always a level of IRI seen in the recipient after transplantation. In fact, any intervention with subsequent tissue trauma, including surgery may trigger hypoxia and inflammation. The period of hypoxia  leads to an anaerobic metabolism in mitochondria with dys- or nonfunction of the respiratory chain and subsequent accumulation of succinate and other metabolites . At repeThese ROS release initiates further downstream sterile inflammation and with damage of the mitochondrial and cellular wall thereby releasing further molecules including danger-associated-molecular patters  and various pro-inflammatory cytokines  . An ongoThe ROS–DAMPS–cytokine cascade instigates further downstream effects in the liver microenvironment, enabling the migration of circulating tumor cells through the sinusoidal endothelial cells (SEC) barrier. In fact, during inflammation, SECs can swell and lose their integrity, thus increasing their permeability. Consequently, tumor cells can migrate, invade and grow. In addition to these mechanisms, hypoxia triggers the release of the hypoxia-inducible-factor, which promotes tumor cell proliferation, migration and angiogenesis, with secretion of vascular endothelial growth factor. Such molecular pathways are of high relevance in liver transplantation, in particular when additional donor and recipient risk factors are involved, such as marginal donors or smaller grafts.Next to the maintenance of liver function and recovery from IRI-injury, mitochondria are essential components to produce enough energy for liver regeneration. Impaired mitochondria trigger delays in liver regeneration particularly described in patients suffering from high inflammation after liver resection or partial transplantation. Delayed hepatocyte cell cycle passages and delayed entrance into the cell cycle were described.The ongoing mitochondrial ROS release and chronic inflammation can also damage healthy cells and their mitochondrial DNA, which may lead to new DNA mutations, and upregulate proto-oncogenes together with a downregulation of tumor-suppressor genes, and hence, the development of new cancers .Centre experience, logistics, medical recipient fitness and the duration of cold ischemia are additional factors with effect on the IRI-severity, the subsequent liver function, regeneration, and recipient recovery. Triggered by an elevated IRI-associated inflammation and related repair, tumor cell spread and growth can also be enhanced during liver transection in the donor and later regeneration . In factMoreover, it has been demonstrated in experimental models that the process of liver splitting induces the release of inflammatory molecules as a result of the parenchyma transection, as observed in models of accelerated liver regeneration such as the Associating Liver Partition and Portal vein Ligation for Staged hepatectomy (ALPPS) technique ,54. HereSimilarly, to the earlier concept of adding a small auxiliary liver graft, known as the concept of APOLT , a technique that was initially performed based on the concept that the residual native recipient liver would support the overall function of the implanted graft until the graft that is, individually, too small would have grown enough .Another interesting surgical technique to discuss is the living donor (LD) resection and partial liver segment 2–3 transplantation with delayed total hepatectomy (RAPID) procedure initially proposed by Königsrainer et al. . AlthougMetabolically less favorable livers are often not easily detected just by quantifying the levels of steatosis or considering the age of the donor. Based on this, there is always a risk of developing significant IRI, contributing to the development of SFSS with impaired liver function . The regMitochondrial dysfunction and advanced inflammation in the recipient are the underlying mechanisms of and ongoing inflammation and downstream microvascular tumor spread and recurrence ,66. SomeProvided that the recipient risk is given with the acceptance of the candidate for transplantation and the same is valid for an available living donor, the remaining “wheel to adjust” appears with the preservation of the organ. Early studies have shown that novel dynamic preservation technologies may reduce IRI, reprogram mitochondria and downregulate the recipient’s immune response ,69,70. TThe second main technique is the hypothermic oxygenated perfusion (HOPE). Pre-cooled (4–10 °C) artificial fluids are recirculated in a pressure controlled system. Interestingly, the HOPE approach delivering high-perfusate oxygen levels under cold conditions was found to reprogram mitochondria and to prevent later IRI-associated inflammation after liver implantation. Various mechanistic and clinical studies exist together with four randomized controlled trials ,80,81,82Although there are currently no clinical studies on the use of machine perfusion for living donor liver grafts, available experimental studies have triggered the enthusiasm to further assess the HOPE concept. Additional evidence comes from living donor kidney transplantation, where injury occurs during graft procurement which could be reversed with hypothermic perfusion, resulting in better early kidney graft function compared to standard cold storage . RecentlThe present study also highlights the importance of a careful donor selection when considering HCC recipients. Machine perfusion could potentially lead to better graft function, less IRI and downregulate immune response, which is directly linked to cancer recurrence ,34. The The present study has several limitations, which should be considered when interpreting the results. The current literature lacks randomized controlled trials, the gold standard study design for comparative studies to provide high-quality evidence. Thus, the best available evidence is based on retrospective observational studies, which are inherently subject to selection bias. These studies consisted of relatively small cohorts and included patients that were treated over a prolonged period of time with more than 20 years. The long period may have introduced additional bias in the context of patient management and modified indications and surgical techniques over time. In addition, donor characteristics  were not reported in the included studies, which could have caused a bias when interpreting the results. Next, it is also important to highlight that two of the included studies did not report the locoregional liver cancer treatment prior to LDLT. Of note, the identification of prognostic factors appeared to be very challenging due to the lack of standardized parameter reporting. We were therefore not able to develop a formal meta-analysis model.The present systematic review has demonstrated that in the setting of LDLT, smaller grafts  could be potentially associated with lower survival rates in HCC recipients, in particular in candidates with tumors outside widely used acceptance criteria for transplantation. A careful donor-recipient selection considering the graft size and liver disease severity with level of portal hypertension for HCC recipients could improve outcomes further.Future research should target IRI-associated inflammation to understand the relationship between graft size and quality, the ability to regenerate and to handle the relatively high portal flow and subsequent tumor recurrence in the setting of LDLT. Novel dynamic preservation techniques could be of benefit and their role should be explored further.The eligibility criteria, methodology and investigated outcome parameters of the current study were underlined in a review protocol. The latter was registered at the International Prospective Register of Systematic Reviews (registration number: CRD42022384456). The methodology used in the present study respected the standards of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .All studies evaluating the outcomes of LDLT comparing the different GRWRs for any adult recipient (aged 18 or older), transplanted for HCC, were considered. Letters, expert opinions, congress abstracts, case series and case reports were excluded. In addition, pediatric transplantation for HCC cases were excluded.LDLT in patients with HCC was considered as the intervention of interest and the different liver GRWRs were evaluated.The inclusion criteria were studies that reported at least one of the following outcomes: 1-, 3- and 5-year recipient survival and DFS rates .A comprehensive search strategy was undertaken based on thesaurus headings, search operators and limits in MEDLINE, EMBASE and Web of Science, and was conducted by two independent authors . The last literature search was conducted on December 15th, 2022. A search algorithm that included the terms “liver transplantation” OR “liver transplant” AND “graft recipient weight ratio” OR “graft size” OR “GRWR” AND “hepatocellular carcinoma” OR “liver cancer” OR “HCC” was performed. No limits were set for the year of the publication, but the language was limited to English. Each study identified and included was searched for additional references to identify any further reports or studies of interest that might have been missed.The assessment of the selected studies using title and abstract was conducted by two reviewers, independently . The full texts of relevant articles were collected and evaluated with the eligibility criteria of this study. study-related data ;baseline demographic and clinical information of the study populations ;outcome data.An electronic data extraction spreadsheet according to Cochrane’s recommendations for intervention reviews was created and was pilot-tested in randomly selected articles and adjusted accordingly. The following information was extracted from each of the included studies by two independent reviewers  to ensure the homogeneity of data collection and to rule out any subjective influence in data collection:Disagreements between the two investigators during this process were resolved following iteration, discussion and consultation with a third, independent senior author (K-H.K.). Complete concordance for all variables was achieved.The quality of included studies was assessed using the Newcastle–Ottawa Scale (NOS) based on selection (four items), comparability (one item) and outcome (three items) . A nine-The potential role of GRWR in the prognosis of patients following LDLT was investigated using 0.8% as the cut-off value based on the reports among available studies. One reviewer (A.P.) independently entered the extracted data into the Review Manager 5.4 software for data synthesis. The entered data were subsequently checked independently by another senior author (A.S.).


American Society for Preventive Cardiology (ASPC) Congress in Arlington, Texas.We hope this finds you all well and that you have enjoyed your summer! It was also great to see so many of you at our recent American Journal of Preventive Cardiology (AJPC) recently received its first impact factor, a very respectable 4.1. This is undoubtedly due to our highly dedicated team of associate editors and general editorial board members, and of course the many experts in preventive cardiology in the United States (US) and beyond who have served as reviewers and/or authors contributing their fine work to the journal. We are also very grateful to the leadership and staff of the American Society for Preventive Cardiology who have supported the journal from its beginning, as well as significantly helping with its dissemination and exposure. We also greatly appreciate our publisher, beginning with Elsevier editor Dr. Koos Admiral in the development of the Journal and more recently Diana Goetz, as well as the many key editorial staff who have worked closely with us. All of you who have contributed countless hours of your time and effort and have believed in the Journal from its inception at the beginning of 2020 should be congratulated for helping to make this happen!As many of you are already aware, the Journal. Moving forward, we hope to consider future special issues – two in progress, one on newer therapies for dyslipidemia and another on women's cardiovascular health, will hopefully be our first such special issues. Another recent initiative is the consideration of joint scientific statements and advisories between the ASPC and other like-minded organizations. One such example is the recent publication of a joint statement on the importance of lipid testing as a performance measure in partnership with the National Lipid Association and its Journal of Clinical Lipidology. The further consideration of ASPC clinical practice statements which advance the field of preventive cardiology, which are also of great interest to the Journal's readership, remain an important priority of the Journal. Finally, the need to further disseminate the Journal to our international audiences remains an important priority, and we hope our overseas readers will further consider their fine work for submission to the Journal.But certainly, it will be important to maintain and hopefully improve the impact factor in years to come. This will require the continued dedication of all involved, and of course our many contributors globally who we of course encourage to think of submitting their high quality work to the AJPC include a short report discussing the topic of very high HDL-C and its potential role in cardiovascular risk assessment, as well as research articles on racial and ethnic differences in NT-pro-BNP levels in US adults, the generalizability of the REDUCE-IT trial in a large diabetes cardiovascular outcomes trial, cardiometabolic predictors of a high risk coronary CT angiography phenotype, and a state-of-the-art review article of the implications of the AHA/ACC/HFSA 2022 Heart Failure Guidelines on the prevention of heart failure.Several articles of interest to point out in this issue of the Journal having a very respectable first impact factor, we hope this will further encourage those you who publish in the preventive cardiology space to consider submitting your fine work for the Journal, as well as to share the opportunity to publish in the Journal with your colleagues. We of course continue to invite you to contact us regarding any suggestions you may have for improving the Journal and continuing to move it forward as the eminent journal in the field.Now with the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.