Estimation of the time of zolpidem intake and differentiation between consumption and external contamination using MALDI-MSI for investigations on single hair samples.

Estimation of drug ingestion time (event time) and distinguishing between drug ingestion and external contamination are important for interpreting hair analysis results in forensics practice. Here, we present a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method for in situ analysis of intact hair. We applied a longitudinal cutting method for a single hair to analysis authentic hair samples from a victim of a drug-facilitated sexual assault (DFSA) case and zolpidem-soaked hair. MALDI-MSI showed that zolpidem-positive segments distributed at 4-6 mm or 6-8 mm from the root in three single hairs of a DFSA victim collected 25 days after the event, at concentrations ranging from 0.1 to 5.7 pg mm-1, in agreement with the results from segmental analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). The estimation of drug intake time was about 20-30 days before sampling, which was consistent with the known time of drug intake. This MALDI-MS method allows imaging analysis of trace substances in a single hair and can realize the intuitive reflection of drug taking time. In addition, zolpidem applied by soaking was mainly distributed on both sides of the longitudinal hair shaft, whereas ingested zolpidem was found only in the middle of the hair shaft of the DFSA victim. The MALDI-MS images of unwashed and washed hair suggested that the amount of externally applied drug was decreased by washing, it was still present on surface layer (cuticle) sides although. Visualization using MALDI-MSI could therefore distinguish between drug ingestion and contamination by reflecting the distribution and deposition site of the drug in hair.

How to sample a seizure plant: the role of the visualization spatial distribution analysis of Lophophora williamsii as an example.

Natural compounds in plants are often unevenly distributed, and determining the best sampling locations to obtain the most representative results is technically challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide the basis for formulating sampling guideline. For a succulent plant sample, ensuring the authenticity and in situ nature of the spatial distribution analysis results during MSI analysis also needs to be thoroughly considered. In this study, we developed a well-established and reliable MALDI-MSI method based on preservation methods, slice conditions, auxiliary matrices, and MALDI parameters to detect and visualize the spatial distribution of mescaline in situ in Lophophora williamsii. The MALDI-MSI results were validated using liquid chromatography-tandem mass spectrometry. Low-temperature storage at -80°C and drying of "bookmarks" were the appropriate storage methods for succulent plant samples and their flower samples, and cutting into 40 µm thick sections at -20°C using gelatin as the embedding medium is the appropriate sectioning method. The use of DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile) as an auxiliary matrix and a laser intensity of 45 are favourable MALDI parameter conditions for mescaline analysis. The region of interest semi-quantitative analysis revealed that mescaline is concentrated in the epidermal tissues of L. williamsii as well as in the meristematic tissues of the crown. The study findings not only help to provide a basis for determining the best sampling locations for mescaline in L. williamsii, but they also provide a reference for the optimization of storage and preparation conditions for raw plant organs before MALDI detection. Key Points: An accurate in situ MSI method for fresh water-rich succulent plants was obtained based on multi-parameter comparative experiments.Spatial imaging analysis of mescaline in Lophophora williamsii was performed using the above method.Based on the above results and previous results, a sampling proposal for forensic medicine practice is tentatively proposed.

MxMPK6-2-mediated phosphorylation enhances the response of apple rootstocks to Fe deficiency by activating PM H+ -ATPase MxHA2.

Iron (Fe) deficiency significantly affects the growth and development, fruit yield and quality of apples. Apple roots respond to Fe deficiency stress by promoting H+ secretion, which acidifies the soil. In this study, the plasma membrane (PM) H+ -ATPase MxHA2 promoted H+ secretion and root acidification of apple rootstocks under Fe deficiency stress. H+ -ATPase MxHA2 is upregulated in Fe-efficient apple rootstock of Malus xiaojinensis at the transcription level. Fe deficiency also induced kinase MxMPK6-2, a positive regulator in Fe absorption that can interact with MxHA2. However, the mechanism involving these two factors under Fe deficiency stress is unclear. MxMPK6-2 overexpression in apple roots positively regulated PM H+ -ATPase activity, thus enhancing root acidification under Fe deficiency stress. Moreover, co-expression of MxMPK6-2 and MxHA2 in apple rootstocks further enhanced PM H+ -ATPase activity under Fe deficiency. MxMPK6-2 phosphorylated MxHA2 at the Ser909 site of C terminus, Thr320 and Thr412 sites of the Central loop region. Phosphorylation at the Ser909 and Thr320 promoted PM H+ -ATPase activity, while phosphorylation at Thr412 inhibited PM H+ -ATPase activity. MxMPK6-2 also phosphorylated the Fe deficiency-induced transcription factor MxbHLH104 at the Ser169 site, which then could bind to the promoter of MxHA2, thus enhancing MxHA2 upregulation. In conclusion, the MAP kinase MxMPK6-2-mediated phosphorylation directly and indirectly regulates PM H+ -ATPase MxHA2 activity at the protein post-translation and transcription levels, thus synergistically enhancing root acidification under Fe deficiency stress.

UQCRFS1 serves as a prognostic biomarker and promotes the progression of ovarian cancer.

UQCRFS1 has been reported to be highly expressed in gastric and breast cancer, but the mechanism remains unclear. The prognosis and biological functions of UQCRFS1 in ovarian cancer (OC) have not been evaluated. The expression of UQCRFS1 in EOC was detected by GEPIA and HPA websites, and the prognosis value was investigated by Kaplan-Meier analysis. Then the correlation between the UQCRFS1 gene and tumor-related signature were analyzed by Spearman correlation analysis and rank sum test. Subsequently, the expression of the UQCRFS1 gene in four ovarian cancer cell lines was detected. A2780 and OVCAR8 with the highest expression of UQCRFS1 were selected in the following biological experiments. Cell proliferation was detected by CCK8 assay, cell cycle and apoptosis were determined by flow cytometry, reactive oxygen species (ROS) production was detected by DCFH-DA, DNA damage gene mRNA expression was analyzed by RT-PCR, and AKT/mTOR pathway protein expression were also examined by western blot after siRNA transfection. We found that UQCRFS1 was high-expression in EOC and associated with poor prognosis. Spearman correlation analysis revealed that the high expression of UQCRFS1 is associated with the cell cycle, apoptosis, oxidative phosphorylation, and DNA damage. Further studies found that knockdown of UQCRFS1 cells reduced cell proliferation, cell cycle arrest at the G1 phase, increased proportion of apoptosis, ROS production, and expression of DNA damage genes, inhibited ATK/mTOR pathway. The study suggested that UQCRFS1 may be a candidated target for diagnosis and treatments in OC.

Inhibit ALDH3A2 reduce ovarian cancer cells survival via elevating ferroptosis sensitivity.

Ovarian cancer (OC) is a malignant gynecologic tumor with high morbidity and mortality. As a newly discovered mode of programmed cell death, ferroptosis has been involved in various pathological processes of kinds of tumors in recent years. Aldehyde dehydrogenase 3 family member A2 (ALDH3A2) catalyzes the oxidation of long-chain aliphatic aldehydes to fatty acid. ALDH3A2 has been shown to be associated with ferroptosis in acute myeloid leukemia (AML), but the mechanism remains unclear. In this study, we analyzed the TCGA and GTEx databases and showed that high ALDH3A2 expression predicted poor prognosis in ovarian cancer. Further studies found that knockout or overexpression of ALDH3A2 correspondingly increased or attenuated the ferroptosis sensitivity of ovarian cancer cells. And sequencing revealed that ALDH3A2 knockout led to the activation of lipid metabolic, GSH metabolic, phospholipid metabolic, and aldehyde metabolic pathways, suggesting that ALDH3A2 induced changes in the sensitivity of ovarian cancer cells to ferroptosis by affecting these metabolic processes. Our results provide a new promising therapeutic strategy for the treatment of OC.

DiatomNet v1.0: A novel approach for automatic diatom testing for drowning diagnosis in forensically biomedical application.

BACKGROUND AND OBJECTIVE: Diatom testing is supportive for drowning diagnosis in forensic medicine. However, it is very time-consuming and labor-intensive for technicians to identify microscopically a handful of diatoms in sample smears, especially under complex observable backgrounds. Recently, we successfully developed a software, named DiatomNet v1.0 intended to automatically identify diatom frustules in a whole slide under a clear background. Here, we introduced this new software and performed a validation study to elucidate how DiatomNet v1.0 improved its performance with the influence of visible impurities. METHODS: DiatomNet v1.0 has an intuitive, user-friendly and easy-to-learn graphical user interface (GUI) built in the Drupal and its core architecture for slide analysis including a convolutional neural network (CNN) is written in Python language. The build-in CNN model was evaluated for diatom identification under very complex observable backgrounds with mixtures of common impurities, including carbon pigments and sand sediments. Compared to the original model, the enhanced model following optimization with limited new datasets was evaluated systematically by independent testing and random control trials (RCTs). RESULTS: In independent testing, the original DiatomNet v1.0 was moderately affected, especially when higher densities of impurities existed, and achieved a low recall of 0.817 and F1 score of 0.858 but good precision of 0.905. Following transfer learning with limited new datasets, the enhanced version had better results, with recall and F1 score values of 0.968. A comparative study on real slides showed that the upgraded DiatomNet v1.0 obtained F1 scores of 0.86 and 0.84 for carbon pigment and sand sediment, respectively, slightly worse than manual identification (carbon pigment: 0.91; sand sediment: 0.86), but much less time was needed. CONCLUSIONS: The study verified that forensic diatom testing with aid of DiatomNet v1.0 is much more efficient than traditionally manual identification even under complex observable backgrounds. In terms of forensic diatom testing, we proposed a suggested standard on build-in model optimization and evaluation to strengthen the software's generalization in potentially complex conditions.

A comprehensive analysis of PANoptosome to prognosis and immunotherapy response in pan-cancer.

PANoptosis, a programmed cell death, shares key characteristics of apoptosis, pyroptosis, and necroptosis. Accumulating evidence suggests that PANoptosis plays a crucial role in tumorigenesis. However, the respective regulation mechanisms in cancer are so far unclear. Using various bioinformatic approaches, we comprehensively analyzed the expression patterns, genetic alterations, prognostic value, and immunological role of PANoptosis genes in pan-cancer. Expression of the PANoptosis gene, PYCARD, was validated based on the Human Protein Atlas database and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). We found that PANoptosis genes were aberrantly expressed in most cancer types, which was consistent with the validation of PYCARD expression. Concurrently, PANoptosis genes and PANoptosis scores were significantly associated with patient survival in 21 and 14 cancer types, respectively. Pathway analysis showed that PANoptosis score was positively correlated with pathways linked to immune and inflammatory responses in pan-cancer, such as IL6-JAK-STAT3 signaling, the interferon-gamma response, and IL2-STAT5 signaling. In addition, the PANoptosis score was significantly correlated with the tumor microenvironment, the infiltration levels of most immune cells (i.e.NK cells, CD8+ T cells, CD4+ T cells, DC cells), and immune-related genes. Furthermore, it was a predictive indicator of immunotherapy response in patients with tumors. These insights substantially improve our understanding of PANoptosis components in cancers and may inspire the discovery of novel prognostic and immunotherapy response biomarkers.

Kinase MxMPK4-1 and calmodulin-binding protein MxIQM3 enhance apple root acidification during Fe deficiency.

Iron (Fe) deficiency is a long-standing issue in plant mineral nutrition. Ca2+ signals and the mitogen-activated protein kinase (MAPK) cascade are frequently activated in parallel to perceive external cues, but their interplay under Fe deficiency stress remains largely unclear. Here, the kinase MxMPK4-1, which is induced during the response to Fe deficiency stress in apple rootstock Malus xiaojinensis, cooperates with IQ-motif containing protein3 (MxIQM3). MxIQM3 gene expression, protein abundance, and phosphorylation level increased under Fe deficiency stress. The overexpression of MxIQM3 in apple calli and rootstocks mitigated the Fe deficiency phenotype and improved stress tolerance, whereas RNA interference or silencing of MxIQM3 in apple calli and rootstocks, respectively, worsened the phenotype and reduced tolerance to Fe deficiency. MxMPK4-1 interacted with MxIQM3 and subsequently phosphorylated MxIQM3 at Ser393, and co-expression of MxMPK4-1 and MxIQM3 in apple calli and rootstocks enhanced Fe deficiency responses. Furthermore, MxIQM3 interacted with the central-loop region of the plasma membrane (PM) H+-ATPase MxHA2. Phospho-mimicking mutation of MxIQM3 at Ser393 inhibited binding to MxHA2, but phospho-abolishing mutation promoted interaction with both the central-loop and C terminus of MxHA2, demonstrating phosphorylation of MxIQM3 caused dissociation from MxHA2 and therefore increased H+ secretion. Moreover, Ca2+/MxCAM7 (Calmodulin7) regulated the MxMPK4-1-MxIQM3 module in response to Fe deficiency stress. Overall, our results demonstrate that MxMPK4-1-MxIQM3 forms a functional complex and positively regulates PM H+-ATPase activity in Fe deficiency responses, revealing a versatile mechanism of Ca2+/MxCAM7 signaling and MAPK cascade under Fe deficiency stress.

Genome-wide identification of apple PPI genes and a functional analysis of the response of MxPPI1 to Fe deficiency stress.

Iron (Fe) deficiency affects plant growth and development. The proton pump interactor (PPI) in plants responds to multiple abiotic stresses, although it has not been well characterized under Fe deficiency stress. In this study, we systematically identified and analyzed the PPI gene family in apple. Three PPI candidate genes were found, and they contained 318-1349 amino acids and 3-7 introns. Under Fe deficiency stress, we analyzed the expression of all the PPI genes in roots of apple rootstock Malus xiaojinensis. Expression of the gene MD11G1247800, designated PPI1, is obviously induced by Fe deficiency treatment in M. xiaojinensis. We first cloned MxPPI1 from M. xiaojinensis and determined its subcellular localization, which indicated that it is localized in the cell membrane and nucleus in tobacco. We found that the level of expression of the MxPPI1 protein increased significantly under Fe deficiency stress in apple calli. Moreover, overexpressing MxPPI1 in apple calli enhanced the activities of ferric chelate reductase and H+-ATPase, H+ secretion, MxHA2 gene expression and total Fe content when compared with the wild type calli. We further found that MxPPI1 interacted with MxHA2 using bimolecular fluorescence complementation and luciferase complementation assays. Overall, we demonstrated that MxPPI1 interacts with MxHA2 to enhance the activity of H+-ATPase to regulate Fe absorption in M. xiaojinensis.

MxMPK4-1 phosphorylates NADPH oxidase to trigger the MxMPK6-2-MxbHLH104 pathway mediated Fe deficiency responses in apple.

Iron (Fe) deficiency is a nutritional stress in plants that commonly occurs in alkaline and calcareous soils. Mitogen-activated protein kinases (MPKs), the terminal player of MAPK cascade, are involved in distinct physiological processes. Once plants suffer from Fe deficiency stress, the mechanism of MPK function remains unclear owing to limited study on the MPK networks including substrate proteins and downstream pathways. Here, the MAP kinase MPK4-1 was induced in roots of Fe efficient apple rootstock Malus xiaojinensis but not in Fe inefficient rootstock Malus baccata under Fe deficiency conditions. Overexpression of MxMPK4-1 in apple calli and apple roots enhanced the responses to Fe deficiency. We found that MxMPK4-1 interacted with NADPH oxidases (NOX)-respiratory burst oxidase homologs MxRBOHD1 and MxRBOHD2, which positively regulated responses to Fe deficiency. Moreover, MxMPK4-1 phosphorylated the C terminus of MxRBOHD2 at Ser797 and Ser906 and positively and negatively regulated NOX activity through these phospho-sites, respectively. When compared with apple calli that overexpressed MxRBOHD2, the coexpression of MxMPK4-1 and MxRBOHD2 prominently enhanced the Fe deficiency responses. We also demonstrated that hydrogen peroxide derived from MxMPK4-1-MxRBOHD2 regulated the MxMPK6-2-MxbHLH104 pathway, illuminating a systematic network that involves different MPK proteins in M. xiaojinensis under Fe deficiency stress.

Comparative analysis of aged documents by desorption electrospray ionization-Mass spectrometry (DESI-MS) imaging.

Document dating is an important and challenging task in the field of forensic science. With the development of analytical techniques, related methods are emerging for document dating studies. Desorption electrospray ionization-mass spectrometry (DESI-MS) is an ambient ionization-MS technique that has been applied in the forensic discrimination of inks. This study focused on utilizing DESI-MS to distinguish between ink entries of different ages. Blue ballpoint ink and black gel ink samples were artificially aged by heat and light exposure and then screened by DESI-MS in the positive ion mode, with a methanol-water-formic acid solution (90:10:0.1%, v/v/v) as the desorption solvent. As a result, the artificially aged ink samples were directly differentiated from the reference ones in the chemical images of age-dependent compounds such as basic dyes or polyethylene glycol derivatives. The amount of ethoxylated fatty amine surfactants in copy paper was also found to decrease after artificial aging. Moreover, this method enabled us to visualize the differences between the composition of naturally aged and fresh ink samples. It has been proven to cause no apparent destruction of the samples. The identification of chronological changes in document materials suggests that the DESI-MS method is a promising approach for relative document dating.

Technical note: Analysis of biological substances in ink fingerprint by desorption electrospray ionization mass spectrometry.

As a custom in China, a visible fingerprint pressed with red seal ink on a signature represents personal confirmation of a document. This custom makes ink fingerprints important evidence for individual identification in the forensic practice of questioned document examination. Recently, forged ink fingerprints using stamps or silica gel fingerprint models have emerged. Consequently, detecting and profiling the pattern of biological substances in visible fingerprints on questioned documents is crucial to prove that a fingerprint was imprinted by its real owner. To solve this problem, a desorption electrospray ionization mass spectrometry (DESI-MS) method was developed to detect the biological substances in ink fingerprints on paper. In the positive ion mode, more signals were detected, but the interference from the seal inks could not be ignored. In the negative ion mode, the ion detected in the sweat latent fingerprints at m/z 187 presented high chemical specificity for MS imaging. It was most likely a borate compound in human sweat. Using this biomarker, the fingerprint pattern was successfully profiled and the hand-imprinted fingerprint was clearly distinguished from the stamp-imprinted one. This method can help in determining the authenticity of ink fingerprints on questioned documents in forensic practice.

Post-mortem evaluation of the pathological degree of myocardial infarction by Fourier transform infrared microspectroscopy.

In clinical and forensic investigations, accurate post-mortem diagnosis of the pathological degree of myocardial infarction (MI) is critical. However, because of the observer variability, the diagnosis cannot be made objectively. Many studies have shown that Fourier transform infrared (FTIR) microspectroscopy is non-invasive, observer-independent, and label-free when analyzing biological tissues. In this study, we used FTIR microspectroscopy in combination with intelligent algorithms to identify the pathological phases in human infarcted cardiac tissues, including ischemia, necrotic, granulation, and fibrotic stages. First, a comparison of infrared spectra corresponding to infarcted tissue pathological categories revealed various spectral properties. The results of unsupervised principal component analysis (PCA) revealed a clear distinction between these four pathological stages and the normal stage. Then, to identify these five stages, an automatic artificial neural network (ANN) classifier was effectively created. Finally, two-dimensional pseudo-color images of two infarcted cardiac tissue sections visualized via the ANN classifier showed great agreement with their histological images. These findings demonstrate that FTIR microspectroscopy has the potential for the post-mortem evaluation of the pathological degree of MI.

MxRop1-MxrbohD1 interaction mediates ROS signaling in response to iron deficiency in the woody plant Malus xiaojinensis.

Iron (Fe) deficiency affects crop production and quality. Rho of plants (ROPs) involves in multiple physiological processes in plants. While it has not been well characterized under Fe deficiency, especially in perennial woody plants. In our study, we cloned ROP homologous gene MxRop1 from Malus xiaojinenesis, then overexpressed it in Arabidopsis, showing enhanced plant tolerance to Fe deficiency, which demonstrated its gene function during this stress. Overexpression of MxRop1 also increased reactive oxygen species (ROS) levels. Moreover, active state of MxRop1 (CA-MxRop1) interacted with N-terminal region of MxrbohD1, one ROS synthesis gene. When MxrbohD1 was overexpressed in apple calli, it showed significantly increased H2O2 content, fresh weight and FCR activity, while ROS inhibitor application dramatically inhibited FCR activity, demonstrating ROS produced by MxrbohD1 regulated Fe deficiency responses. Furthermore, using Agrobacterium rhizogenes transformation, MxrbohD1 was overexpressed in apple roots, with increased expression of Fe deficiency-induced genes and increased root FCR activity. Under Fe deficiency, it exhibited slight leaf yellowing phenotype. Co-expression of CA-MxRop1 and MxrbohD1 significantly induced ROS generation. Finally, we proposed that MxRop1 interacted with MxrbohD1 to modulate ROS mediated Fe deficiency adaptive responses in Malus xiaojinensis, which will provide a guidance of cultivation of Fe-deficiency tolerant apple plant.

MxMPK6-2-bHLH104 interaction is involved in reactive oxygen species signaling in response to iron deficiency in apple rootstock.

Iron (Fe) is a trace element necessary for plant growth. Many land plants have evolved a set of mechanisms associated with the Fe absorption process to deal with the problem of insufficient Fe supply in the soil. During Fe absorption, reactive oxygen species (ROS) can be used as a signal to initiate a response to stress caused by Fe deficiency. However, the molecular mechanisms underlying the involvement of ROS in the Fe deficiency stress response remains unclear. In this study, we have identified a kinase, MxMPK6-2, from Malus xiaojinensis, an apple rootstock that is highly efficient at Fe absorption. MxMPK6-2 has been shown to be responsive to ROS signals during Fe deficiency, and MxMPK6-2 overexpression in apple calli enhanced its tolerance to Fe deficiency. We further screened for proteins in the Fe absorption pathway and identified MxbHLH104, a transcription factor which interacts with MxMPK6-2. MxbHLH104 can be phosphorylated by MxMPK6-2 in vivo, and we confirmed that its phosphorylation increased Fe absorption in apple calli under Fe deficiency, with the presence of ROS promoting this process. Overall, we have demonstrated that MxMPK6-2 is responsive to ROS signaling during Fe deficiency, and is able to control its response by regulating MxbHLH104.

The operating regularity of the stepper motor of thermal inkjet printers and its application in the China national accreditation service 2017ZO146 altered documents identification proficiency test.

With the continuous innovation of inkjet printing technology, the methods of using inkjet printers for falsification have also constantly evolved, leading to increasing difficulties in identifying printing alterations made by the same inkjet printer. This paper mainly studies the operating regularity of the stepper motor of inkjet printers to determine the operating mechanism of inkjet printers and thus to identify whether documents had been tampered with. To detect the operating track of the stepper motor, 154 documents printed by 22 different brands and models of thermal inkjet printers were studied according to the periodic morphological characteristics of ink marks and the track of the stepper motor during different printing processes. As a result: ① the maximum gauge of 22 printheads was found to be different and ② the different distribution of ink marks were mainly affected by the direction and speed of the stepper motor. The track of the stepper motor was able to be determined by the periodic morphological characteristics of the ink marks combined with the maximum gauges, and could be used to judge how many times a document was printed. The observed regularities were evaluated by the CNAS 2017ZO146 test, a national proficiency test in China. In conclusion, using the summarized operating regularities of the stepper motor, it is possible to identify printing alterations made by the same thermal inkjet printer.

Determination of causes of death via spectrochemical analysis of forensic autopsies-based pulmonary edema fluid samples with deep learning algorithm.

This study investigated whether infrared spectroscopy combined with a deep learning algorithm could be a useful tool for determining causes of death by analyzing pulmonary edema fluid from forensic autopsies. A newly designed convolutional neural network-based deep learning framework, named DeepIR and eight popular machine learning algorithms, were used to construct classifiers. The prediction performances of these classifiers demonstrated that DeepIR outperformed the machine learning algorithms in establishing classifiers to determine the causes of death. Moreover, DeepIR was generally less dependent on preprocessing procedures than were the machine learning algorithms; it provided the validation accuracy with a narrow range from 0.9661 to 0.9856 and the test accuracy ranging from 0.8774 to 0.9167 on the raw pulmonary edema fluid spectral dataset and the nine preprocessing protocol-based datasets in our study. In conclusion, this study demonstrates that the deep learning-equipped Fourier transform infrared spectroscopy technique has the potential to be an effective aid for determining causes of death.

Ethylene Response Factors MbERF4 and MbERF72 Suppress Iron Uptake in Woody Apple Plants by Modulating Rhizosphere pH.

Iron (Fe) deficiency limits the yield of fruit trees. When subjected to Fe deficiency, H+ secretion increases in the rhizosphere of dicotyledonous plants and pH decreases. This leads to the acidification of the soil and promotes Fe3+ to Fe2+ conversion, which plants can better uptake. This study investigated the relationship between two inhibitory transcription factors (ethylene response factors MbERF4 and MbERF72) and the H+-ATPase gene MbHA2. Two species of apple woody plants were studied: the Fe-inefficient Malus baccata and the Fe-efficient Malus xiaojinensis. Yeast one-hybrid and electrophoretic mobility shift assays showed that both MbERF4 and MbERF72 bind to the GCC cassette (AGCCGCC) of the MbHA2 promoter. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that MbERF4 interacts with MbERF72. Furthermore, ß-glucuronidase and luciferase reporter assays showed that the MbERF4- and MbERF72-induced repression of MbHA2 expression is synergistic. Virus-induced gene silencing of MbERF4 or MbERF72 increased MbHA2 expression, and thus lowered the rhizosphere pH in M. baccata. Consequently, the high expressions of MbERF4 and MbERF72 induced by Fe deficiency contributed to the Fe sensitivity of M. baccata. Moreover, the low expressions of MxERF4 and MxERF72 contributed to the Fe-deficiency tolerance of M. xiaojinensis via different binding conditions to the HA2 promoter. In summary, this study identified the relationship of two inhibitory transcription factors with the H+-ATPase gene and proposed a model in which ERF4 and ERF72 affect the rhizosphere pH in response to Fe deficiency.

Genome-Wide Identification and Characterization of ABC Transporters in Nine Rosaceae Species Identifying MdABCG28 as a Possible Cytokinin Transporter linked to Dwarfing.

ATP-binding cassette (ABC) transporters constitute a large, diverse, and ubiquitous superfamily that is involved in a broad range of processes. The completion of genome sequencing provides an opportunity to understand the phylogenetic history of the ABC transporter superfamily among Rosaceae species. This study identified a total of 1323 ABC transporter genes from nine Rosaceae genomes: 191 from Malus domestica, 174 from Pyrus communis, 138 from Prunus persica, 118 from Prunus avium, 141 from Prunus dulcis, 122 from Fragaria vesca, 98 from Rubus occidentalis, 162 from Prunus mume, and 179 from Rosa chinensis. Their chemical characterization, phylogenetic analysis, chromosomal localization, gene structure, gene duplication, and tissue-specific expression were studied. Their subcellular localization, transmembrane structures, and protein motifs were predicted. All the ABC transporter genes were grouped into eight subfamilies on the basis of their phylogenetic relationships and structural features. Furthermore, cis-element and expression analysis of 10 potential phytohormone transporters in MdABCG subfamily genes were also performed. Loss of the W-box in the promoter region of MdABCG28 was found to reduce the gene expression level and was linked to the dwarfing phenotype in apple rootstocks. MdABCG28 overexpression promoted shoot growth of atabcg14 mutants in Arabidopsis.

Biochemical detection of fatal hypothermia and hyperthermia in affected rat hypothalamus tissues by Fourier transform infrared spectroscopy.

It is difficult to determinate the cause of death from exposure to fatal hypothermia and hyperthermia in forensic casework. Here, we present a state-of-the-art study that employs Fourier-transform infrared (FTIR) spectroscopy to investigate the hypothalamus tissues of fatal hypothermic, fatal hyperthermic and normothermic rats to determine forensically significant biomarkers related to fatal hypothermia and hyperthermia. Our results revealed that the spectral variations in the lipid, protein, carbohydrate and nucleic acid components are highly different for hypothalamuses after exposure to fatal hypothermic, fatal hyperthermic and normothermic conditions. In comparison with the normothermia group, the fatal hypothermia and hyperthermia groups contained higher total lipid amounts but were lower in unsaturated lipids. Additionally, their cell membranes were found to have less motional freedom. Among these three groups, the fatal hyperthermia group contained the lowest total proteins and carbohydrates and the highest aggregated and dysfunctional proteins, while the fatal hypothermia group contained the highest level of nucleic acids. In conclusion, this study demonstrates that FTIR spectroscopy has the potential to become a reliable method for the biochemical characterization of fatal hypothermia and hyperthermia hypothalamus tissues, and this could be used as a postmortem diagnostic feature in fatal hypothermia and hyperthermia deaths.