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Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ijpp.2023.03.003, Objective: To evaluate the presence of Dicrocoelium sp. in a child from a Late Antique funerary context from Cantabrian Spain and discuss whether the infection is true infection or pseudoparasitosis. Materials: Four skeletons, including one from a 5–7 year old child, have been analysed from the archaeological site of El Convent ́on, dated between the sixth and seventh centuries AD. Methods: The paleoparasitological study was conducted through the analysis of soil samples from different parts of the skeleton and funerary context using the rehydration, homogenization, and micro-sieving method, and visualized through brightfield microscopy. Results: A soil sample from the pelvic region tested positive for Dicrocoelium sp. (possibly D. dendriticum). Conclusions: The child was infected with Dicrocoelium dendriticum, which based on archaeological and historical contexts may be related to hygiene or dietary behaviour. Significance: We present one of the few cases of the identification of a Dicrocoelidae parasite directly associated with a human skeleton that provides historical knowledge of a zoonotic disease. Limitations: The diagnosis of a zoonosis through the identification of ancient parasites is complex. In addition, Dicrocoelium sp. in association with skeletal human remains is rare due to the potential low prevalence of this parasite. Suggestions for Further Research: Highlight the importance of paleoparasitological analysis to link parasitic infection diseases with socioeconomic issues by using funerary contexts with skeletal remains., European Union’s Horizon 2020 the Marie Skłodowska-Curie TRAUMOBITA 895712

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Membrane contact sites (MCS) are regions where two membranes of different organelles are close but not fused; they coordinate non-vesicular communication between organelles and are involved in a wide variety of physiological functions, including membrane lipid homeostasis. Amongst proteins localized at MCS are those containing a lipid transport domain known as synaptotagmin-like mitochondrial-lipid binding protein (SMP), being the mammalian Extended Synaptotagmins, the yeast Tricalbins and the plant Synaptotagmin 1 (SYT1) the best SMP proteins characterized so far. They are all localized at endoplasmic reticulum-plasma membrane contact sites (ER-PM CS). We have carried outin-silicogenome-wide identification of genes encoding SMP proteins in Arabidopsis and tomato. We have identified the plant exclusive NTMC2T5 proteins as ER-chloroplast CS components which make them extremely interesting as the route for lipid trafficking into and out of chloroplasts remains unknown. Additionally,NTMC2T5over-expressions caused a significant clustering of chloroplast around nucleus. Moreover, SYT6, NTMC2T6 and TEX2 have been identified as ER-Trans-Golgi Network CS proteins. These proteins associated between them and with the exocytosis related proteins VAMP721 and VAMP727. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies.HIGHLIGHTPlant exclusive lipid transport proteins were identified at membrane contact sites. SYT6, TEX2 and NTMC2T6 proteins are localized at ER-TGN. NTMC2T5 proteins are localized at ER-Chloroplast and induced chloroplast-nucleus clustering.

El hallazgo de parásitos procedentes de contextos arqueológicos nos permite conocer las condiciones socio-económicas y los hábitos alimentarios de las poblaciones pasadas, aportando una visión novedosa en el estudio de dichas sociedades. Pese a su probado interés en la contribución al conocimiento de los estudios arqueológicos, antropológicos y biológicos, apenas se han llevado a cabo investigaciones paleoparasitológicas en España. Este trabajo presenta los resultados de los análisis paleoparasitológicos desarrollados sobre restos esqueletizados de ocho individuos procedentes de la necrópolis romana de Dianium (Dénia, Alicante, España), fechada entre los siglos III a V d.C. Este análisis se ha realizado mediante visualización por microscopía óptica de campo claro. Los resultados han evidenciado la presencia de Ascaris lumbricoides en uno de los individuos. Estos resultados se han integrado con los hallazgos paleopatológicos y antropológicos. La presencia de este parásito se vincula a la falta de higiene, derivada de contaminación ambiental y alimentaria.

Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane., Science Advances, 8 (46), ISSN:2375-2548

Age-at-death estimation from skeletal remains typically utilizes the roughness of pubic symphysis articular surfaces. This study presents a new quantitative method adapting a tool from geometric morphometrics, bandpass filtering of partial warp bending energy to extract only age-related changes of the surfaces. The study sample consisted of 440 surface-scanned symphyseal pubic bones from men between 14 and 82 years of age, which were landmarked with 102 fixed and surface semilandmarks. From the original sample, 371 specimens within Procrustes distance of 0.05 of the side-specific average were selected. For this subsample, age was correlated with total bending energy (calculated as summed squared partial warps amplitudes) for a wide range of plausible bandpass filters. For our subsample’s 188 right-side surfaces, the correlation between age and bandpass filtered versions of bending energy peaks relatively sharply at r = −0.648 for ages up through 49 years against the first seven partial warp amplitudes only. The finding for left symphyses is similar. The results demonstrate that below the age 50, the symphyseal surface form changes most systematically related to age may be best detected by a lowpass-filtered version of bending energy: signals at the largest geometric scales of roughness rather than its full spectrum. Combining this method with information from other skeletal features could further improve age-at-death estimation based on the symphyseal pubic surface.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff–Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Rare earth elements induce ER membrane remodeling and increase ER–PM connectivity in a process that involves phosphoinositide-associated reorganization of synaptotagmin-tethering complexes., In plant cells, environmental stressors promote changes in connectivity between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM). Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in interorganelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+- and lipid-binding protein 1 (CLB1/SYT7). This complex is enriched at ER–PM contact sites (EPCSs), has slow responses to changes in extracellular Ca2+, and displays severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositide species at the PM.

Objective To interpret traumatic cranial lesions on two individuals buried at the Neolithic necropolis of Campo de Hockey, Spain. Materials Skeletal remains and grave goods associated with two individuals recovered in good condition from a single grave. Methods Crania were macroscopically and radiographically analysed. Results Of all the individuals recovered from this site, only two present perimortem skull injuries. Fractures were located on the right side of the skulls, one on the frontal bone and the other on the parietal bone. Conclusions The individuals present signs of interpersonal violence. In both cases, the injuries are perimortem and may have contributed to the deaths of the individuals. Significance The identification of interpersonal violence in the two individuals buried in the tomb, alongside the archaeological context of the burials, provides unique insight into Neolithic burial practices in the Iberian Peninsula and the role that paleopathology can play in the recognition of social complexity. Limitations The skeletal remains of the two individuals were fragmented, rendering the identification of perimortem lesions difficult. Suggestions for further research New imaging techniques, including CT-scan, will provide more detailed analyses.

Plant SYT1 transports lipids between endoplasmic reticulum and plasma membrane in response to stress. The protein flexibility and lipid-binding specificity determine SYT1 function at molecular level., Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

Forensic humanitarian work occurring both in the judicial context, and in the novel extrajudicial model implemented in Colombia as a result of the peace treaty signed between the Colombian government and the FARC, requires the creation of new demographic standards for the Colombian population in the context of the forensic identification. In this paper we describe a new forensic demographic standard, consisting of a quantitative technique to estimate the sex of human remains resulting from the armed conflict, by measuring canine teeth in a sample of 162 adult individuals that belong to the contemporary Colombian skeletal reference collection that belongs to the National Institute of Legal Medicine and Forensic Sciences in Bogotá D.C. The results of the new technique indicate that the sex was correctly estimated in 82.1% of the cases when using upper canines, and it was correctly estimated in 77.8% of the cases when using lower canines. In the context of human rights violations, the fact that the Colombian conflict has resulted in about 120,000 disappeared, has led to the implementation of transitional justice measures based on a comprehensive justice system of truth, reparation and non-repetition. From the forensic arena, we propose a new methodology for sex estimation as a way to contribute to the process of justice, truth and reparation.

The bony symphyseal surface is an important trait for age-at-death estimation from human skeletal remains. In this paper, we demonstrate for the first time that the outline of the human symphyseal surface is sexually dimorphic and that it changes with age. We present a geometric morphometric analysis based on a sample of 323 symphyseal pubic bones from males and females in the age range of 14 to 82 years. These bones were surface-scanned and the resulting surface models were measured along the ventral and dorsal borders of the symphyseal surface using two fixed and 36 curve semilandmarks. Our findings imply that age-related changes in the outline of the symphyseal surface differ between the sexes. According to our results, age explains 5% of total shape variation in females, but less than 1% in males. These findings for the outline, could potentially complement existing sex and age-at-death estimation methods based on other features of the bony pubic surface.

17 pags., 7 figs., 1 tab., Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER., This work was funded by grants from Agencia Estatal de Investigación (AEI, Spain) and Fondo Europeo de Desarrollo Regional (European Union) (BIO2017-89523-R to A Albert). A Albert and D Siliqi thank ALBA (beamline XALOC) and Diamond Light Source (beamline B21, proposal MX21741) for granting access to the synchrotron radiation source. We thank Nathan Cowieson and Charlotte JC Edwards-Gayle from Diamond Light Source for their assistance in the preparation of the SAXS experiments

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein–protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

The interorganelle communication mediated by membrane contact sites (MCSs) is an evolutionary hallmark of eukaryotic cells. MCS connections enable the nonvesicular exchange of information between organelles and allow them to coordinate responses to changing cellular environments. In plants, the importance of MCS components in the responses to environmental stress has been widely established, but the molecular mechanisms regulating interorganelle connectivity during stress still remain opaque. In this report, we use the model plant Arabidopsis thaliana to show that ionic stress increases endoplasmic reticulum (ER)-plasma membrane (PM) connectivity by promoting the cortical expansion of synaptotagmin 1 (SYT1)-enriched ER-PM contact sites (S-EPCSs). We define differential roles for the cortical cytoskeleton in the regulation of S-EPCS dynamics and ER-PM connectivity, and we identify the accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the PM as a molecular signal associated with the ER-PM connectivity changes. Our study highlights the functional conservation of EPCS components and PM phosphoinositides as modulators of ER-PM connectivity in eukaryotes, and uncovers unique aspects of the spatiotemporal regulation of ER-PM connectivity in plants.

Sex determination is one of the essential steps when it comes to establishing an individual's biological profile. It is important in both archaeology and forensic studies. The sacrum is not generally conserved, but in cases where it is, it can be used for determination of the sex of skeletal remains. Furthermore, the sacrum is not a commonly studied bone and has not been studied in a Spanish population. For this study, measurements of 170 sacra of individuals from the contemporary osteological collection of San José from Granada including only the adults were taken. Measurements based on the anatomical regions of the sacra were established in order to obtain some regression formulas to determine sex. Our results show that the Superior Transverse Line and Right Lateral Sacral Crest are the most dimorphic structures, achieving a 74% rate of correct classification of sex in a univariate analysis. In multivariate analysis, when the mentioned variables were combined, an 81.41% rate of correct classification was achieved. Our results show that our method can be applied with other methods at the same time to determine the sex of individuals in forensic and archaeological contexts.

Mutations in the Arabidopsis TETRATRICOPEPTIDE THIOREDOXIN-LIKE 1 (TTL1) gene cause reduced tolerance to osmotic stress evidenced by an arrest in root growth and root swelling, which makes it an interesting model to explore how root growth is controlled under stress conditions. We found that osmotic stress reduced the growth rate of the primary root by inhibiting the cell elongation in the elongation zone followed by a reduction in the number of cortical cells in the proximal meristem. We then studied the stiffness of epidermal cell walls in the root elongation zone of ttl1 mutants under osmotic stress using atomic force microscopy. In plants grown in control conditions, the mean apparent elastic modulus was 448% higher for live Col-0 cell walls than for ttl1 (88.1 ± 2.8 vs. 16.08 ± 6.9 kPa). Seven days of osmotic stress caused an increase in the stiffness in the cell wall of the cells from the elongation zone of 87% and 84% for Col-0 and ttl1, respectively. These findings suggest that TTL1 may play a role controlling cell expansion orientation during root growth, necessary for osmotic stress adaptation.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

23 pags., 6 figs., Endoplasmic reticulum–plasma membrane contact sites (ER–PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER–PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER–PM tether that also functions in maintaining PM integrity. The ER–PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work, This work was supported by the Ministerio de Economıa y Competitividad, co-financed by the European Regional Development Fund (grant no. BIO2017-82609-R to M.A.B.), the Ministerio de Ciencia, Innovacion y Universidades (grant no. PGC2018-098789-B-I00 to N.R.-L.) UMA-FEDER (grant UMA18-FEDERJA-154 to N.R.-L.), and the Marie SkłodowskaCurie actions (grant no. H2020-655366-IIF- PLICO to M.A.B. and N.R.-L.). N.R.L. was supported by the Ramon y Cajal program RYC-2013-12699 (MINECO, Spain). J.P.-S. and S.G.-H. were funded by the Ministerio de Economıa y Competitividad in Formacion del Personal Investigador Fellowship (grant no. BES-2012-052324) and (PRE2018- 085284), respectively. R.P.H. and J.A.N. received support from the Biotechnology and Biological Sciences Research Council (BBSRC, UK) in the form of an Institute Strategic Programme Grant (grant no. BBS/E/C/000I0420). J.L. is supported by the Program of Introducing Talents of Discipline to Universities (111 Project, grant no. B13007). A.P.M. and J.P.-S. were supported by the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences), Chinese 1000 Talents Program. A.R. was supported by the Natural Sciences and Engineering Research Council of Canada (NSERCDiscovery Grant no. RGPIN-2019-05568). Support was also provided by AEI/FEDER, UE (grant nos. BIO2016-79187-R and PID2019-106987RB-I00 to J.P.-S.) and by the European Research Council under the European Union’s Seventh Framework Programme (grant no. FP7/2007-2013)/ERC grant agreement no. 742985 to J.F. and T-Rex (project number 682436 to D.V.D.).

SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

The enzymatic steps involved inl-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions inNicotiana benthamianaand Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, andl-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP andl-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complementedvtc2null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes inN. benthamianasupported the roleof VTC2as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 andl-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

© 2019 The Authors., FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid‐1,4‐lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9‐specific CArG box was identified in the promoters of these genes., This work was supported by grants BIO2013-44199-R, AGR12- 40066-CO2-02, and RTI2018-099797-B-100 (MINECO, Spain), and the European Union’s H2020 Programme (GoodBerry; grant number 679303) and ERC-2014-StG638134. DP and SO acknowledge the support by Spanish Ministry of Science and Innovation (Ramon and Cajal contracts, RYC2011-09170 and RYC2013-12699).

Introduction: Preoperative treatment and adequate surgery increase local control in rectal cancer. However, modalities and indications for neoadjuvant treatment may be controversial. Aim of this study was to assess the trends of preoperative treatment and outcomes in patients with rectal cancer included in the Rectal Cancer Registry of the Spanish Associations of Surgeons. Method: This is a STROBE-compliant retrospective analysis of a prospective database. All patients operated on with curative intention included in the Rectal Cancer Registry were included. Analyses were performed to compare the use of neoadjuvant/adjuvant treatment in three timeframes: I)2006–2009; II)2010–2013; III)2014–2017. Survival analyses were run for 3-year survival in timeframes I-II. Results: Out of 14, 391 patients, 8871 (61.6%) received neoadjuvant treatment. Long-course chemo/radiotherapy was the most used approach (79.9%), followed by short-course radiotherapy ± chemotherapy (7.6%). The use of neoadjuvant treatment for cancer of the upper third (15-11 cm) increased over time (31.5%vs 34.5%vs 38.6%, p = 0.0018). The complete regression rate slightly increased over time (15.6% vs 16% vs 18.5%; p = 0.0093); the proportion of patients with involved circumferential resection margins (CRM) went down from 8.2% to 7.3%and 5.5% (p = 0.0004). Neoadjuvant treatment significantly decreased positive CRM in lower third tumors (OR 0.71, 0.59–0.87, Cochrane-Mantel-Haenszel P = 0.0008). Most ypN0 patients also received adjuvant therapy. In MR-defined stage III patients, preoperative treatment was associated with significantly longer local-recurrence-free survival (p < 0.0001), and cancer-specific survival (p < 0.0001). The survival benefit was smaller in upper third cancers. Conclusion: There was an increasing trend and a potential overuse of neoadjuvant treatment in cancer of the upper rectum. Most ypN0 patients received postoperative treatment. Involvement of CRM in lower third tumors was reduced after neoadjuvant treatment. Stage III and MRcN + benefited the most.

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.

Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.

In plant cells, environmental stressors induce changes in the cytosolic concentration of calcium ([Ca2+]cyt) that are transduced by Ca2+-sensing proteins. To confer specificity to the stress signaling response, [Ca2+]cytsensing must be tightly regulated in space and time; the molecular mechanisms that restrict the localization and dynamics of Ca2+sensors in plants, however, are largely unknown. In this report, we identify a putative Ca2+-sensitive complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+-dependent lipid binding protein (CLB1), which is enriched at ER-PM contact sites (EPCS) and relocalizes in response to Rare Earth Elements (REEs)-induced endocytosis. Our results show that endocytosed REEs influence cytosolic Ca2+signaling, as indicated by the activation of the Ca2+/Calmodulin-based ratiometric sensor GCaMP3, and promote the cytoskeleton-dependent accumulation of ER-PM contact sites at the cell cortex. Based on these results, we propose that the EPCS-localized SYT1/SYT5/CLB1 complex is part of an evolutionarily conserved and spatially regulated Ca2+-responsive mechanism that control cER-PM communication during stress episodes.

One of the most important adaptations of seagrasses during sea colonization was the capacity to grow at the low micromolar nitrate concentrations present in the sea. In contrast to terrestrial plants that use H+ symporters for high-affinity NO3&minus, uptake, seagrasses such as Zostera marina L. use a Na+-dependent high-affinity nitrate transporter. Interestingly, in the Z. marina genome, only one gene (Zosma70g00300.1, NRT2.1) is annotated to this function. Analysis of this sequence predicts the presence of 12 transmembrane domains, including the MFS domains of the NNP transporter family and the &ldquo, nitrate signature&rdquo, that appears in all members of the NNP family. Phylogenetic analysis shows that this sequence is more related to NRT2.5 than to NRT2.1, sharing a common ancestor with both monocot and dicot plants. Heterologous expression of ZosmaNRT2-GFP together with the high-affinity nitrate transporter accessory protein ZosmaNAR2 (Zosma63g00220.1) in Nicotiana benthamiana leaves displayed four-fold higher fluorescence intensity than single expression of ZosmaNRT2-GFP suggesting the stabilization of NRT2 by NAR2. ZosmaNRT2-GFP signal was present on the Hechtian-strands in the plasmolyzed cells, pointing that ZosmaNRT2 is localized on the plasma membrane and that would be stabilized by ZosmaNAR2. Taken together, these results suggest that Zosma70g00300.1 would encode a high-affinity nitrate transporter located at the plasma membrane, equivalent to NRT2.5 transporters. These molecular data, together with our previous electrophysiological results support that ZosmaNRT2 would have evolved to use Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize marine environments.

The interorganelle communication mediated by membrane contact sites (MCSs) is an evolutionary hallmark of eukaryotic cells. MCS connections enable the nonvesicular exchange of information between organelles and allow them to coordinate responses to changing cellular environments. In plants, the importance of MCS components in the responses to environmental stress has been widely established, but the molecular mechanisms regulating interorganelle connectivity during stress still remain opaque. In this report, we use the model plant Arabidopsis thaliana to show that ionic stress increases endoplasmic reticulum (ER)–plasma membrane (PM) connectivity by promoting the cortical expansion of synaptotagmin 1 (SYT1)-enriched ER–PM contact sites (S-EPCSs). We define differential roles for the cortical cytoskeleton in the regulation of S-EPCS dynamics and ER–PM connectivity, and we identify the accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] at the PM as a molecular signal associated with the ER–PM connectivity changes. Our study highlights the functional conservation of EPCS components and PM phosphoinositides as modulators of ER–PM connectivity in eukaryotes, and uncovers unique aspects of the spatiotemporal regulation of ER–PM connectivity in plants.

Throughout evolution, a number of animals including humans have lost the ability to synthesize ascorbic acid (ascorbate, vitamin C), an essential molecule in the physiology of animals and plants. In addition to its main role as an antioxidant and cofactor in redox reactions, recent reports have shown an important role of ascorbate in the activation of epigenetic mechanisms controlling cell differentiation, dysregulation of which can lead to the development of certain types of cancer. Although fruits and vegetables constitute the main source of ascorbate in the human diet, rising its content has not been a major breeding goal, despite the large inter- and intraspecific variation in ascorbate content in fruit crops. Nowadays, there is an increasing interest to boost ascorbate content, not only to improve fruit quality but also to generate crops with elevated stress tolerance. Several attempts to increase ascorbate in fruits have achieved fairly good results but, in some cases, detrimental effects in fruit development also occur, likely due to the interaction between the biosynthesis of ascorbate and components of the cell wall. Plants synthesize ascorbate de novo mainly through the Smirnoff-Wheeler pathway, the dominant pathway in photosynthetic tissues. Two intermediates of the Smirnoff-Wheeler pathway, GDP-D-mannose and GDP-L-galactose, are also precursors of the non-cellulosic components of the plant cell wall. Therefore, a better understanding of ascorbate biosynthesis and regulation is essential for generation of improved fruits without developmental side effects. This is likely to involve a yet unknown tight regulation enabling plant growth and development, without impairing the cell redox state modulated by ascorbate pool. In certain fruits and developmental conditions, an alternative pathway from D-galacturonic acid might be also relevant. We here review the regulation of ascorbate synthesis, its close connection with the cell wall, as well as different strategies to increase its content in plants, with a special focus in fruits.

Introdução: A capacidade laboral se converte em um instrumento de amplo alcance que faz referência às condições de saúde de trabalhadores. Objetivos: Avaliar o Índice de Capacidade para o Trabalho (ICT) e as condições de saúde de trabalhadores com deficiência fí­sica de uma Instituição de Ensino Superior em João Pessoa/PB. Material e métodos: Trata-se de um estudo observacional, transversal e de natureza quantitativa, avaliado através do ICT para o Trabalho de 13 trabalhadores com deficiência fí­sica (TCDF) e 13 sem deficiência fí­sica (TSDF). Resultados: Constataram-se diferenças significativas (p = 0,0076), identificando que os TSDF apresentaram escore médio no ICT mais elevado (45,15 ± 4,25) caracterizando a capacidade laboral como ótima, e os TCDF (40,62 ± 3,66) com ICT boa. Quando comparado o ICT com o fator do trabalhador ter ou não deficiência fí­sica, obteve-se (p = 0,021). No grupo de TCDF as lesões prevaleceram na coluna, nas pernas e pés, enquanto que no grupo de TSDF predominou as lesões na coluna. Conclusão: Verificou-se que o estado de saúde está associado com o ICT e que os TCDF mesmo apresentando maiores comprometimentos em relação às alterações musculoesqueléticas são capazes de realizar atividades laborais com independência e autonomia no seu posto de trabalho.Palavras-chave: avaliação da capacidade de trabalho, saúde do trabalhador, pessoas com deficiência.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. BR perception at the plasma membrane initiates a series of phosphorylation events enabling the nuclear accumulation and activity of the key transcription factors BZR1 and BES1. Previous studies support that all BR signaling components form an interconnected pathway, although it is not known how these proteins are brought together for the prompt signal transduction from the plasma membrane to the nucleus. Here we report that plant-specific Tetratricopeptide Thioredoxin- Like (TTL) proteins are positive regulators of BR signaling that function as scaffold for the BR signaling components in Arabidopsis. TTL3 directly interacts with BZR1 and the phosphatase BSU1, and associates in vivo with most core components involved in BR signaling, with the exception of the BAK1 coreceptor. TTL3 is mainly localized in the cytoplasm, and BR treatment increases its localization at the plasma membrane. In addition, the expression of TTL3 strengthens the association of BR-signaling components BSK1 and BZR1 at the plasma membrane. Consistent with a role in BR signaling, mutations in TTL3, and related TTL1 and TTL4 genes cause reduced BR responses, and these defects that highly enhanced in a triple ttl1 ttl3 ttl4 mutant. We propose a novel mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex with the goal of ensuring dephosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1. Interestingly, this novel TTL scaffold model for BR signaling resembles that of Wnt signaling in metazoans, in which TTL proteins would act similar to Axin1, optimizing signaling efficiency of the cascade by promoting the assembly of the signaling complex at the plasma membrane.

Despite the high mortality that have been associated with pregnancy and childbirth in past populations, few cases of the remains of pregnant women burials are described in the bioarchaeological reports. This paper is focused on the case of a double inhumation found in the Medieval Muslim Necropolis (11

Sex determination of unknown individuals is one of the primary goals of Physical and Forensic Anthropology. The adult skeleton can be sexed using both morphological and metric traits on a large number of bones. The human pelvis is often used as an important element of adult sex determination. However, studies carried out about the pelvic bone in subadult individuals present several limitations due the absence of sexually dimorphic characteristics. In this study, we analyse the sexual dimorphism of the immature pubis and ischium bones, attending to their shape (Procrustes residuals) and size (centroid size), using an identified sample of subadult individuals composed of 58 individuals for the pubis and 83 for the ischium, aged between birth and 1year of life, from the Granada osteological collection of identified infants (Granada, Spain). Geometric morphometric methods and discriminant analysis were applied to this study. The results of intra- and inter-observer error showed good and excellent agreement in the location of coordinates of landmarks and semilandmarks, respectively. Principal component analysis performed on shape and size variables showed superposition of the two sexes, suggesting a low degree of sexual dimorphism. Canonical variable analysis did not show significant changes between the male and female shapes. As a consequence, discriminant analysis with leave-one-out cross validation provided low classification accuracy. The results suggested a low degree of sexual dimorphism supported by significant sexual dimorphism in the subadult sample and poor cross-validated classification accuracy. The inclusion of centroid size as a discriminant variable does not imply a significant improvement in the results of the analysis. The similarities found between the sexes prevent consideration of pubic and ischial morphology as a sex estimator in early stages of development. The authors suggest extending this study by analysing the different trajectories of shape and size in later ontogeny between males and females.

Summary The receptacle of the strawberry (Fragaria × ananassa) fruit accounts for the main properties of the ripe fruit for human consumption. As it ripens, it undergoes changes similar to other fruits in sugar : acid ratio, volatile production and cell wall softening. However, the main regulators of this process have not yet been reported. The white stage marks the initiation of the ripening process, and we had previously reported a peak of expression for a FaGAMYB gene. Transient silencing of FaGAMYB using RNAi and further determination of changes in global gene expression by RNAseq, and composition of primary and secondary metabolites have been used to investigate the role played by this gene during the development of the receptacle. Down-regulation of FaGAMYB caused an arrest in the ripening of the receptacle and inhibited colour formation. Consistent with this, several transcription factors associated with the regulation of flavonoid biosynthetic pathway showed altered expression. FaGAMYB silencing also caused a reduction of ABA biosynthesis and sucrose content. Interestingly, exogenous ABA application to the RNAI-transformed receptacle reversed most defects caused by FaGAMYB down-regulation. The study assigns a key regulatory role to FaGAMYB in the initiation of strawberry receptacle ripening and acting upstream of the known regulator ABA.

Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca2+ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.

Radiology has demonstrated great utility for age estimation, but most of the studies are based on metrical and morphological methods in order to perform an identification profile. A simple image analysis-based method is presented, aimed to correlate the bony tissue ultrastructure with several variables obtained from the grey-level histogram (GLH) of computed tomography (CT) sagittal sections of the pubic symphysis surface and the pubic body, and relating them with age. The CT sample consisted of 169 hospital Digital Imaging and Communications in Medicine (DICOM) archives of known sex and age. The calculated multiple regression models showed a maximum R (2) of 0.533 for females and 0.726 for males, with a high intra- and inter-observer agreement. The method suggested is considered not only useful for performing an identification profile during virtopsy, but also for application in further studies in order to attach a quantitative correlation for tissue ultrastructure characteristics, without complex and expensive methods beyond image analysis.

Background: Suppression of Fra a gene expression leads to down-regulation of color-producing flavonoid biosynthesis in strawberry. Results: Fra proteins can bind natural flavonoids, which induce conformational changes in conserved loop regions. Conclusion: Fra a proteins control flavonoid biosynthesis through binding to metabolic intermediates. Significance: PR-10 proteins may play a role in the control of secondary metabolism through binding of metabolites to their ligand-binding cavities., Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

The methodology used to identify individuals in forensic anthropology requires a minimum degree of preci- sion and accuracy and should be based on identified and representative samples. Achievement of these objectives in infant skeletons is hampered by the scarcity of appropriate samples. The dental age estimation methods of Liversidge et al. (Am J Phys Anthropol 90: 307-313, 1993 )a nd Deutsch et al. (Growth 49: 207-17, 1985 )w ere applied to the Granada osteological collection of identified infants (Granada, Spain) in order to evaluate its applicability in a Mediterranean population. Significant differences were found between the estimated and real ages in both cases. Based on the measurements obtained in 140 fetuses and infants, new regression formulas were developed to estimate age from the metric study on deciduous teeth. Independent functions are provided for each deciduous maxillary and mandibular tooth in each sex, along with the margin of error (95 % confidence interval). These formulas appear to offer one of the best methods available for estimating the age of Mediterranean infants in forensic anthropology settings.