Acid Transport Research Articles

Streptococcus canis transcriptomic modifications in host cell entry environments of human keratinocytes

BackgroundStreptococcus canis is a commensal bacterium in companion animals. This microorganism can infect humans who have been in deep contact with or bitten by pet dogs, suggesting that the skin/soft tissue is one of infection entry sites. To understand pathological process in human cells, we aimed to determine S. canis transcriptomic changes in invasive environments of human keratinocytes.MethodsWe selected one isolate from candidates with whole-genome sequences, based on re-obtained cell invasion ability (CIA) data into human keratinocytes along with bacterial cytotoxicity. RNA-sequencing was conducted for the samples at baselines and 2 h/5 hr post-inoculation using NovaSeq 6000. Global/differential gene expression analyses [principal component analysis (PCA)/k-means clustering analysis/differentially expressed gene (DEG) analyses] were performed. We classified DEGs into their functional categories. To validate transcriptomic results, we did quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays.ResultsFU1 isolate was selected from seven candidates, based on re-obtained CIA data with less cytotoxicity. Total read bases of 6.17–9.02 Gbp were obtained by RNA-sequencing. PCA and k-means clustering analysis indicated clustering according to their inoculation times. Volcano plots and Venn diagrams revealed that S. canis invasion into keratinocytes produced altered distributions of many genes. Gene ontology enrichment analysis showed most of the gene expressions were downregulated. DEG functional analysis showed the downregulated DEGs belonging to energy production and conversion/carbohydrate transport and metabolism/amino acid transport and metabolism/nucleotide transport and metabolism, with the upregulated DEGs belonging to transcription. qRT-PCR assays for downregulated/upregulated expressions of four genes (pgk–slo/opuAA–kdpB) validated transcriptomic results.ConclusionOur observations suggest that S. canis can downregulate its metabolism-associated gene expressions in human keratinocyte environments. The observed gene expression changes can imply the latent infection in human cells. Further investigation is needed to elucidate the underlying mechanisms for the latent infection.

Coupled Geomechanics and Chemistry Model for Acid Stimulation of Naturally Fractured Carbonate Reservoirs

Summary Treatments of naturally fractured carbonate formations typically involve acid injection when damage of natural fractures (NFs) occurs mechanically and chemically. Coupled modeling of both mechanisms is often neglected. We build a quantitative model of damageable reservoir, where the permeability depends on the shear and tensile activation of NFs, as well as acid transport and reaction. Simulations demonstrate model applicability to NF acidizing and acid fracturing. Particularly, we show that strong pressure drawdowns in tight carbonates result in steep production declines because of NF pinching.

Identification of potential pathogenic targets and survival strategies of Vibrio vulnificus through population genomics.

Vibrio vulnificus, a foodborne pathogen, has a high mortality rate. Despite its relevance to public health, the identification of virulence genes associated with the pathogenicity of currently known clinical isolates of V. vulnificus is incomplete and its synergistic pathogenesis remains unclear. Here, we integrate whole genome sequencing (WGS), genome-wide association studies (GWAS), and genome-wide epistasis studies (GWES), along with phenotype characterization to investigate the pathogenesis and survival strategies of V. vulnificus. GWAS and GWES identified a total of six genes (purH, gmr, yiaV, dsbD, ramA, and wbpA) associated with the pathogenicity of clinical isolates related to nucleotide/amino acid transport and metabolism, cell membrane biogenesis, signal transduction mechanisms, and protein turnover. Of these, five were newly discovered potential specific virulence genes of V. vulnificus in this study. Furthermore, GWES combined with phenotype experiments indicated that V. vulnificus isolates were clustered into two ecological groups (EGs) that shared distinct biotic and abiotic factors, and ecological strategies. Our study reveals pathogenic mechanisms and their evolution in V. vulnificus to provide a solid foundation for designing new vaccines and therapeutic targets.

Insight into the mechanism of H+-coupled nucleobase transport

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

A data-driven model to estimate the pore volume to breakthrough for carbonate acidizing

This research investigates the impact of different rock, acid, and reaction dynamic properties on the pore volume to breakthough (PVBT) at different acid injection rates using in-house developed two-scale continuum simulation model. We analyzed the parameters relation and developed a reliable machine learning model to accurately predict the PVBT at similar range of investigated parameters. In the simulation, it was found that different acid concentrations result in the same optimum injection velocity but at large contrast in PVBT between low and high acid concentration. However, other parameters such as diffusion coefficient and reaction rate exhibited an inverse PVBT behavior across optimum injection velocity due to change in acid transport and reaction behavior. After that, different reliable machine learning algorithms were employed to predict the optimum PVBT for carbonates matrix acidizing. The utilized machine learning models undergone multiple optimizations and comparison to obtain the most accurate prediction performance. The artificial neural network model with 2 hidden layers outperforms the other optimizations with 11.27% estimation error, 0.96 R2 and 0.98 correlation coefficient for the testing data set. Finally, an empirical correlation was developed to accurately estimate PVBT at a low cost and very short time compared to lab experiments and numerical simulation models. The novelty of this research stems from examining PVBT curves behavior by varying five matrix acidizing parameters independently, analyzing the correlation between these parameters and developing machine learning model for handy and reliable optimum PVBT estimation.

Mineralogy Impact on Acid Fracture Design in Naturally Fractured Carbonates.

The impacts of a natural fracture on the propagation of a hydraulic fracture has been extensively studied in the past decade. However, there were only minor attempts to understand the impact of natural fractures on acid distribution during acid fracturing. This study provides an understanding of the impact of natural fractures on acid fracturing designs in limestone and dolomite formations. A built-in-house model that solves fracture propagation, acid transport and dissolution, heat transfer, and reservoir productivity was utilized. A sensitivity analysis was conducted to investigate the impact of temperature, injection rate, and acid type. One of the significant differences between limestone and dolomite is the dissolution profile in the hydraulic fracture. The maximum dissolution happens close to the wellbore in limestone, but far from the wellbore in dolomite at low acid temperatures. Also, the hydraulic fracture in dolomite could have lower conductivity (i.e., permeability) compared to the intersecting natural fractures, which is not usually the case in limestone. Hence, the productivity enhancement by acid fracturing in limestone formation is higher than in dolomite. In general, natural fractures reduce productivity, and their negative impact is more significant in dolomite. The sensitivity study showed that the acid temperature significantly impacts fractured well productivity of dolomite, especially at high reservoir permeability where higher dissolution is desired. It is observed that the optimum injection rate that maximizes productivity is much higher in limestone than in dolomite. Also, acid retardation does not positively impact the productivity of dolomite compared to limestone; however, high viscosity might be needed to keep the hydraulic fracture open. This is the first study to reveal mineralogy's impact on acid fracture performance in naturally fractured carbonates.

The release of FB1-induced heterophil extracellular traps in chicken is dependent on autophagy and glycolysis

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin produced by Fusarium, poses a great threat to the poultry industry. It was reported that extracellular traps could be induced by FB1 efficiently in chickens. However, the relevance of autophagy and glycolysis in FB1-triggered heterophil extracellular trap (HET) formation is unclear. In this study, immunostaining revealed that FB1-induced HETs structures were composed of DNA coated with histones H3, and elastase, and that heterophils underwent LC3B-related autophagosome formation assembly driven by FB1. Western blotting showed that FB1 downregulated the phosphorylated phosphoinositide 3-kinase3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin complex 1 (mTORC1) axis and raised the AMP-activated kinase α (AMPKα) activation protein. Furthermore, rapamycin- and 3-Methyladenine (3-MA)-treatments modulated FB1-triggered HET formation according to the pharmacological analysis. Further studies on energy metabolism showed that glucose/lactate transport and glycolysis inhibitors abated FB1-induced HETs. These results showed that FB1-induced HET formation might interact with the autophagy process and relied on glucose/monocarboxylic acid transporter 1 (MCT1) and glycolysis, reflecting chicken's early innate immune responses against FB1 intake.

The protective role of adiponectin in restoring spermatogenesis in mice fed on methionine-choline deficient diet

Introduction and Aim: The process of spermatogenesis requires a continuous supply of nutrients for producing normal sperms. Methionine and choline are considered important amino acids in transport of energy substrates, redox balance, and in DNA methylation while the protein adiponectin is known for its anti-inflammatory and anti-apoptotic function. In this study, we aimed to investigate the effect of methionine and choline on mice testis when fed with a methionine choline deficient diet (MCDD) as well as the protective effect of adiponectin on mice testis. Materials and Methods: This study comprised of 25 mice, which were divided into three groups: Control (n=5); M1 (n=10 that were fed with MCDD for three weeks) and M2 (n=10 that were fed with MCDD for three weeks and in the third week were administered with daily injections of adiponectin for one week). Results: The present study revealed MCDD caused a significant reduction in body weight and increase in the incidence of apoptosis where P value was 0.001; caspase-3 expression was higher than that of Bcl2 in group M1. Adiponectin therapy in the last week showed the potential to restore body weight, reduce apoptotic rate, and the expression of Bcl2 which was higher than that of caspase-3. Conclusion: A diet lacking in methionine and choline not only significantly reduced body weight in mice, but also increased the apoptosis rate of spermatogenic cells. Adiponectin therapy in MCDD fed mice reduced the apoptotic rate and restored spermatogenesis.

Abscisic acid affects the floret numbers of inflorescence by regulating indole-3-acetic acid transport and accumulation in Lavandula angustifolia

Lavender flower essential oil had been used for a variety of therapeutic and cosmetic purposes. Previous studies mainly focused on essential oil composition and extraction methods, ignoring the factors that affected the production of essential oils, such as the floret number. This study aims to get a deep insight into the mechanism of floret number differences. Hormone analysis showed that there was a positive correlation between abscisic acid (ABA) content and the number of florets, while indole-3-acetic acid (IAA) was negatively correlated with floret numbers. RNA-Seq results showed that 1 035 differentially expressed genes (DEG) can be identified, of which 19 genes were significantly enriched in the carotenoid biosynthesis pathway. Common network analysis showed that the expression of the key genes of ABA pathway metabolism was negatively related to the DEG of IAA, especial IAA18. Exogenous IAA significantly inhibited the number of lavender florets and affected the expression of ABA metabolism genes. The results might be useful for a better understanding of the molecular mechanism of floret number differences.

Evidence for a Prehypertensive Water Dysregulation Affecting the Development of Hypertension: Results of Very Early Treatment of Vasopressin V1 and V2 Antagonism in Spontaneously Hypertensive Rats.

In addition to long-term regulation of blood pressure (BP), in the kidney resides the initial trigger for hypertension development due to an altered capacity to excrete sodium and water. Betaine is one of the major organic osmolytes, and its betaine/gamma-aminobutyric acid transporter (BGT-1) expression in the renal medulla relates to interstitial tonicity and urinary osmolality and volume. This study investigated altered water and sodium balance as well as changes in antidiuretic hormone (ADH) activity in female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats from their 3–5 weeks of age (prehypertensive phase) to SHR’s 28–30 weeks of age (established hypertension-organ damage). Young prehypertensive SHRs showed a reduced daily urine output, an elevated urine osmolarity, and higher immunostaining of tubule BGT-1, alpha-1-Na-K ATPase in the outer medulla vs. age-matched WKY. ADH circulating levels were not different between young prehypertensive SHR and WKY, but the urine aquaporin2 (AQP2)/creatinine ratio and labeling of AQP2 in the collecting duct were increased. At 28–30 weeks, hypertensive SHR with moderate renal failure did not show any difference in urinary osmolarity, urine AQP2/creatinine ratio, tubule BGT-1, and alpha-1-Na-K ATPase as compared with WKY. These results suggest an increased sensitivity to ADH in prehypertensive female SHR. On this basis, a second series of experiments were set to study the role of ADH V1 and V2 receptors in the development of hypertension, and a group of female prehypertensive SHRs were treated from the 25th to 49th day of age with either V1 (OPC21268) or V2 (OPC 41061) receptor antagonists to evaluate the BP time course. OPC 41061-treated SHRs had a delayed development of hypertension for 5 weeks without effect in OPC 21268-treated SHRs. In prehypertensive female SHR, an increased renal ADH sensitivity is crucial for the development of hypertension by favoring a positive water balance. Early treatment with selective V2 antagonism delays future hypertension development in young SHRs.

The Function of Amino Acid Transporters in Plant

Amino acids are a kind of nutrient element necessary for plant growth and development. Through the participation of amino acid transporters, the transport of amino acids between aboveground and underground parts of plants and the loading of amino acids from xylem to phloem can be completed. At present, many genes encoding plant amino acid transporters have been isolated and cloned, but little is known about the functions of different amino acid transporters and their effects on plants. Therefore, this paper reviewed the role of amino acid transporters in plants from three aspects: the classification, isolation, and cloning of amino acid transporters genes and the functional research of amino acid transporters, to provide references for the utilization of amino acid transporters in plants.

MO038: Mineral bone density assessment in patients affected by cystinuria

Abstract BACKGROUND AND AIMS Cystinuria is a rare genetic disease characterized by impaired tubular transport of cystine and other dibasic amino acids that leads to cystine renal calculi. Although being a rare condition, cystinuria is the most frequent monogenic cause of nephrolithiasis accounting for 1% of kidney stones in adults and 8% in children with an average prevalence of 1 in 7000 births. Up to date, clinical features of cystinuria are believed to be attributable only to nephrolithiasis although cystinuric patients may present other comorbidities (chronic kidney disease, progression toward end-stage kidney disease and hypertension). All these features may be partially explained by recurrent nephrolithiasis. There are currently no data on bone features of patients with cystinuria. Data on a murine model demonstrated the presence of reduced bone mineral density irrespective of the presence or absence of uremia, suggesting bone metabolic abnormalities in cystinuria even in the absence of reduced glomerular filtration rate (GFR). Our aim is to characterize bone mineral density (BMD) in patients affected by cystinuria. METHOD The present study included 13 patients affected by cystinuria followed at the Rare Kidney Diseases outpatient clinic of our Institution. All eligible patients were more than 18 years of age with an estimated GFR ≥15 mL/min/1.73 m2 and provided written informed consent. Patients who did not sign informed consent, pregnant women, patients undergoing chronic hemodialysis or with a previous kidney transplant were excluded. History and laboratory data were collected during scheduled outpatient visits from September 2021 to December 2021, and all patients underwent a dual-energy X-ray absorptiometry (DEXA). One sample t-tests were used to assess whether BMD Z-scores were significantly different from 0. RESULTS Patients’ characteristics are summarized in Table 1. Of the enrolled patients, six (46%) were female and four of them (67%) were in menopause, four (31%) had active nephrolithiasis described as at least two episodes in the last 3 months, and two (15%) had a self-reported diagnosis of osteoporosis. Moreover, six patients (46%) had a positive history for bone fractures that were all self-reported as traumatic. Femoral Z-scores were significantly lower than 0 overall (P = 0.015) and across femoral sites, whereas lumbar Z-scores, although low on average, did not meet statistical significance (Figure 1). Interestingly, reduced BMD was very prevalent, with 10 patients (77%) showing osteopenia and 3 (23%) showing osteoporosis. CONCLUSION Our data show a high prevalence of osteopenia and osteoporosis in cystinuric patients, especially when considering that our population was rather young and with a normal or moderately impaired kidney function. Of further interest appears also the differential distribution of decreased BMD that seems to be affecting the femur rather than the lumbar spine. To our knowledge, these are the first data assessing the prevalence and the characteristics of BMD disorder in cystinuric patients.

Role of cannabinoids and vitamin E analogues in macrophages foam cells formation

Recently, macrophages foam cells have gained attention in cases of electronic‐cigarette or vaping product use‐associated lung injury (EVALI). An increased number of foam cells was detected in bronchoalveolar lavages and lungs after inhaling e‐cigarette smoke from products containing cannabinoids and the vitamin E analogue, alpha‐tocopherol acetate (αTA), that was present as vape‐cartridge additive. Increased levels of intact αTA are reportedly detected in EVALI indicating that conversion of αTA to αT may less occur in lungs upon inhaling vapors from e‐cigarettes containing αTA.HypothesisWhereas the molecular mechanisms by which macrophages foam cells are formed after vaping are unclear, it appears possible that cannabinoid receptors play a role, as lung‐resident macrophages express cannabinoid receptors. Dysregulation of cannabinoid receptors by αTA and cannabinoids may induce the CD36 scavenger receptor/fatty acids transporter (CD36/FAT) and/or reduce the ATP binding cassette transporters A1 and G1 (ABCA1/G1) expression leading to lipid accumulation. Therefore, we analyzed the regulatory effects on lipid homeostasis of natural alpha‐tocopherol (RRR‐αT), natural RRR‐αTA (αTAn) and synthetic racemic all‐rac‐αTA (αTAr) with and without co‐treatment with cannabinoids (cannabinol (CBD), Δ9‐tetrahydrocannabinol (THC)) in THP‐1 monocytes and macrophages (differentiated for 48 h with 100 ng/ml phorbol‐12‐myristate‐13‐acetate (PMA)).ResultsQuantification of lipids in macrophages indicated that more lipids accumulate with αT, less with αTAn and αTAr, whereas control alpha‐tocopheryl phosphate (αTP) clearly inhibited. Co‐treatment with THC increased lipid accumulation with αT, αTAn as well as with CBD, whereas αTP again clearly inhibited. In THP‐1 monocytes, CD36 scavenger receptor was stimulated with αTAn and THC and with co‐treatment with THC and αTAn or CBD. In THP‐1 macrophages, CD36 was only up‐regulated by CBD and co‐treatment with αTAn or αTAr reduced it. In THP‐1 monocytes, cell cycle progression was only affected by CBD (increased G1, decreased S) and co‐treatment with αTAn and αTAr restored it. In macrophages, no significant toxicity was measured with lactate dehydrogenase (LDH) release assay by these treatments at 24 h, 48 h and 72 h at αTA concentrations below 50 μM.SummaryAlthough the mechanisms by which intact αTA may affect lungs in cases of EVALI are still unclear it may disrupt the pulmonary surfactant, act as linactant, and/or dysregulate lipid homeostasis and contribute to formation of macrophages foam cells and inflammation. Lipid accumulation in monocytes/macrophages and foam cells formation were modulated by cannabinoids and αTA but the involvement of lipid homeostasis genes such as CD36/FAT and ABCA1/G1 remains to be further elucidated. A detailed analysis of the molecular mechanisms by which foam cells are formed will not only be important to understand EVALI but also for atherosclerosis and non‐alcoholic steatohepatitis (NASH).

Investigating Differential Expressed Genes of Limosilactobacillus reuteri LR08 Regulated by Soybean Protein and Peptides.

Soybean protein and peptides have the potential to promote the growth of Lactobacillus, but the mechanisms involved are not well understood. The purpose of this study is to investigate differentially expressed genes (DEGs) of Limosilactobacillus reuteri (L. reuteri) LR08 responding to soybean protein and peptides using transcriptome. The results showed that both digested protein (dpro) and digested peptides (dpep) could enhance a purine biosynthesis pathway which could provide more nucleic acid and ATP for bacteria growth. Moreover, dpep could be used instead of dpro to promote the ABC transporters, especially the genes involved in the transportation of various amino acids. Interestingly, dpro and dpep played opposite roles in modulating DEGs from the acc and fab gene families which participate in fatty acid biosynthesis. These not only provide a new direction for developing nitrogen-sourced prebiotics in the food industry but could also help us to understand the fundamental mechanism of the effects of dpro and dpep on their growth and metabolisms and provides relevant evidence for further investigation.

RNA-Seq Transcriptomic Analysis of Green Tea Polyphenols Modulation of Differently Expressed Genes in Enterococcus faecalis Under Bile Salt Stress.

Enterococcus faecalis (E. faecalis) belongs to lactic acid bacteria which can be used as a probiotic additive and feed, bringing practical value to the health of humans and animals. The prebiotic function of tea polyphenols lays a foundation for green tea polyphenols (GTP) to repair the adverse changes of E. faecalis under stress conditions. In this study, RNA-sequence analysis was used to explore the protective effect of GTP on E. faecalis under bile salt stress. A total of 50 genes were found to respond to GTP under bile salts stress, containing 18 up-regulated and 32 down-regulated genes. The results showed that multiple genes associated with cell wall and membrane, transmembrane transport, nucleotide transport and metabolism were significantly differentially expressed (P < 0.05), while GTP intervention can partly alleviate the detrimental effects of bile salt on amino acid metabolism and transport. The present study provides the whole genome transcriptomics of E. faecalis under bile salt stress and GTP intervention which help us understand the growth and mechanism of continuous adaptation of E. faecalis under stress conditions.

The Gastrodia menghaiensis (Orchidaceae) genome provides new insights of orchid mycorrhizal interactions

BackgroundTo illustrate the molecular mechanism of mycoheterotrophic interactions between orchids and fungi, we assembled chromosome-level reference genome of Gastrodia menghaiensis (Orchidaceae) and analyzed the genomes of two species of Gastrodia.ResultsOur analyses indicated that the genomes of Gastrodia are globally diminished in comparison to autotrophic orchids, even compared to Cuscuta (a plant parasite). Genes involved in arbuscular mycorrhizae colonization were found in genomes of Gastrodia, and many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids. The highly expressed genes for fatty acid and ammonium root transporters suggest that fungi receive material from orchids, although most raw materials flow from the fungi. Many nuclear genes (e.g. biosynthesis of aromatic amino acid L-tryptophan) supporting plastid functions are expanded compared to photosynthetic orchids, an indication of the importance of plastids even in totally mycoheterotrophic species.ConclusionGastrodia menghaiensis has the smallest proteome thus far among angiosperms. Many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids.

Competing Transport of Malonic and Acetic acids across Commercial and Modified RALEX AMH Anion-Exchange Membranes

Competing Transport of Malonic and Acetic acids across Commercial and Modified RALEX AMH Anion-Exchange Membranes

Establishment of Immortalized Yak Ruminal Epithelial Cell Lines by Lentivirus-Mediated SV40T and hTERT Gene Transduction.

Yak is a unique species of cattle that is adapted to the harsh natural environment of the Qinghai-Tibet Plateau. Research on the function of the yak rumen is limited to animal experiments, and the cell molecular mechanism is very limited. The high cost of isolation and culture of adult yak rumen epithelial cells (YRECs), low success rate, and limited cell life limit the scope of long-term physiological functions and nutrient absorption mechanisms of yak rumen epithelium in vitro studies. This study aimed to explore the isolation and immortal culture methods of primary YRECs and establish a new cell line model for studying cell molecular mechanisms. The human telomerase reverse transcriptase gene (hTERT) and simian virus 40 large T antigen (SV40T) were transferred into primary YDECs using mammalian gene expression lentiviral vectors. The immortalized cell line (SV40T-YREC-hTERT) retains the morphological and functional characteristics of primary cells. The epithelial cell marker protein cytokeratin 18 of the immortalized cell lines was positive, and the cell proliferation and karyotype were normal. The SV40T and hTERT genes were successfully transferred into immortalized cell lines and maintained high expression. Simultaneously, the immortalized cell lines had normal function of short-chain fatty acid (SCFA) transport and absorption, and the immortalized yak rumen epithelial cell lines were successfully established. In addition, the transepithelial electrical resistance value gradually increased with culture time, and the permeability of epithelial cells decreased by culturing epithelial cells in Transwell culture chambers. Transmission electron microscopy demonstrated the submicroscopic structure of cells in the integrity barrier model and established the YREC barrier model in vitro.

Dietary L-Carnitine Affects Leukocyte Count and Function in Dairy Cows Around Parturition.

In early lactation, an energy deficit leading to a negative energy balance (NEB) is associated with increased susceptibility to disease and has been shown to be an important factor during transition in dairy cows. L-carnitine as a key factor in the mitochondrial transport of fatty acids and subsequently for β-oxidation and energy release is known to modulate mitochondrial biogenesis and thus influence metabolism and immune system. In the current study, we characterized hematological changes around parturition and investigated the potential effects of dietary L-carnitine supplementation on immune cell functions. For this approach, dairy cows were assigned either to a control (CON, n = 30) or an L-carnitine group [CAR, n = 29, 25 g rumen-protected L-carnitine per cow and day (d)]. Blood samples were taken from d 42 ante partum (ap) until d 110 post-partum (pp), with special focus and frequent sampling from 0.5 to72 h post-calving to clarify the impact of L-carnitine supplementation on leukocyte count, formation of reactive oxygen species (ROS) in polymorphonuclear cells (PMN) and peripheral mononuclear cells (PBMC) and their phagocytosis activity. Blood cortisol concentration and the capacity of PBMC proliferation was also investigated. All populations of leukocytes were changed during the peripartal period, especially granulocytes showed a characteristic increase up to 4 h pp. L-carnitine supplementation resulted in increased levels of eosinophils which was particularly pronounced one day before to 4 h pp, indicating a possible enhanced support for tissue repair and recovery. Non-supplemented cows showed a higher phagocytic activity in PBMC as well as a higher phagocytic capacity of PMN during the most demanding period around parturition, which may relate to a decrease in plasma levels of non-esterified fatty acids reported previously. L-carnitine, on the other hand, led to an increased efficiency to form ROS in stimulated PMN. Finally, a short period around calving proved to be a sensitive period in which L-carnitine administration was effective.

Mix-and-match: an improved, fast and accessible protocol for hypocotyl micrografting of Arabidopsis seedlings with systemic ACC responses as a case study

BackgroundGrafting is a technique widely used in horticulture that also has been applied in agriculture. In plant physiology, grafting facilitates the elucidation of mechanisms underlying growth and developmental processes, through the construction of chimeric plants with organs of different genotypes. Despite its small size, the model species Arabidopsis thaliana is very amenable for grafting, which can be useful to investigate transport of nutrients, amino acids or secondary metabolites between different tissues, or to investigate developmental processes depending on root-to-shoot communication, such as shoot branching, root and shoot plasticity upon shade avoidance, or disease resistance. Nevertheless, grafting protocols are usually technically challenging and training is required to achieve a reasonable success rate. Additionally, specialized tools and equipment are often needed, such as chips to accommodate the grafted plantlets or collars to maintain the contact between root and shoot.ResultsIn this methodology paper, we provide a fast, easy, accessible, and specialized equipment-free protocol that enables high success ratios. Critical steps and notes are detailed, easing the implementation of the procedure for non-trained researchers. An example of the protocol application by three independent non-trained researchers shows that this method allows to achieve a 90–100% of grafting efficiency after 6 days post-grafting recovery. In addition, the grafting of Col-0 with the acs8x mutant, depleted in 1-aminocyclopropane-1-carboxylic acid (ACC), the biosynthetic precursor of the phytohormone ethylene, provides an example of the application of this optimized protocol, showing the suitability of the process to study long-distance transport processes.ConclusionsWe present an optimized protocol for hypocotyl grafting of 4-day-old Arabidopsis thaliana seedlings. The combination of conditions yields a grafting success of 90–100% and provides an easy and accessible methodology, reducing the time frame, and without the necessity of acquiring specialized equipment. The presented protocol is simple, fast and highly efficient, easing the inclusion of hypocotyl grafting assays in any research project. In addition, the description of the protocol is detailed to a level ensuring that even non-trained researchers, are sufficiently prepared to adopt the grafting methodology.