Pi Treatment Research Articles

Effects of inorganic phosphate on stem cells isolated from human exfoliated deciduous teeth

Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (Pi) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). SHEDs were treated with Pi. Cell cycle progression and apoptosis were examined using flow cytometry analysis. Osteo/odontogenic and adipogenic differentiation were analyzed using alizarin red S and oil red O staining, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. Pi increased the late apoptotic cell population while cell cycle progression was not altered. Pi upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. Pi-induced mineralization was reversed by pretreatment of cells with Foscarnet, or p38 inhibitor. Pi treatment inhibited adipogenic differentiation as determined by decreased PPARγ expression and reduced intracellular lipid accumulation. Bioinformatic analysis of gene expression profiles demonstrated several involved pathways, including PI3K/AKT, MAPK, EGFR, and VEGF signaling. In conclusion, Pi enhanced osteo/odontogenic but inhibited adipogenic differentiation in SHED.

Above- and Below-Ground Interactions and Interspecific Relationships in Wheat/Maize Systems

Above- and below-ground interactions play a crucial role in achieving higher yields in intercropping systems. Nonetheless, it remains unclear how these interactions impact intercropping crop growth and regulate interspecific relationships. This study aimed to quantify the impact of above- and below-ground interactions on crop yield by determining the dynamics of dry matter accumulation, photosynthetically active radiation (PAR) transmittance, and leaf area index (LAI) in intercropped wheat and maize. Three below-ground intensities were set for an intercropping system: no root separation (CI: complete interaction below ground), 48 μm nylon mesh separation (PI: partial interaction below ground), and 0.12 mm plastic sheet separation (NI: no interaction below ground). Two densities were set for maize: low (45,000 plants hm−2) and high (52,500 plants hm−2). At the same time, corresponding monoculture treatments were established. The grain yields in the CI and PI treatments were, on average, 23.7% and 13.7% higher than those in the NI treatment at high and low maize densities, respectively. Additionally, the grain yield for high density was 12.3% higher than that of low density in the CI treatment. The dry matter accumulation of intercropped wheat under the CI and PI treatments was, on average, 9.1%, 14.5%, and 9.0% higher than that in the NI treatment at the flowering, filling, and maturity stages, respectively. The dry matter accumulation of intercropped maize at the blister, milk, and physiological maturity stages increased by 41.4%, 32.1%, and 27.8%, respectively, under the CI treatment compared to the NI treatment. The PAR transmittance and LAI of maize at the V6 stage were significantly increased by increasing the intensity of below-ground interactions. This study showed that complete below-ground interaction contributed to a significant increase in the competitiveness of intercropped wheat with respect to maize (Awm) under the high-density maize treatment, especially at the filling stage of wheat. Moreover, the CI treatment enhanced the recovery effects of maize (Rm) after wheat harvesting. Increasing the intensity of below-ground interactions can significantly enhance the Awm and Rm in intercropping systems, favoring the accumulation of crop dry matter mass and light energy utilization to increase system yields.

Prospective multicenter study to describe the prevalence, outcomes, and management of phosphate disorders in intensive care patients: Study protocol for part B of the international GUTPHOS study

BackgroundAberrations in blood phosphate (Pi) levels, whether presenting as hypo- or hyperphosphatemia, appear to be associated with clinical complications and adverse outcomes in patients admitted to an intensive care unit (ICU). However, the prevalence of Pi disorders and the association with subsequent factors and organ failures leading to death in ICU patients are poorly described. Despite endeavors to understand the etiology and treatment of low Pi levels from systematic reviews and meta-analyses, the literature lacks comprehensive guidance for managing hypophosphatemia. Hyperphosphatemia, on the other hand, appears to be associated with higher mortality among critically ill patients, yet its prevalence among ICU patients, particularly following phosphate repletion, remains unknown. The present study aims to investigate the prevalence of Pi abnormalities upon ICU admission and their incidence during the first week of ICU stay, the factors associated with Pi alterations, and the effect of phosphate repletion on the normalization of Pi levels, and its associations with clinical outcomes. MethodsThis multicentre, prospective, non-interventional cohort study will include at least 1000 consecutive adult ICU patients (≥18 years) as part B of the GUTPHOS study. Sites are eligible if an anticipated minimal inclusion of 50 eligible patients during eight weeks from January 2024 until June 2024 and daily phosphate measurements during the first seven days of ICU stay are expected. All consecutive adult patients admitted to a participating ICU during the recruitment period, lasting up to eight weeks, or up to 120 patients if enrollment reaches that limit earlier, will be included. Study parameters include study site characteristics, patient demographics, daily assessment of Pi levels, Pi-related treatment, feeding details, renal replacement therapy details, the incidence of refeeding-associated hypophosphatemia and administered medication (during the first seven calendar days of ICU stay). There will be a follow-up period of a maximum of 90 days to document 28- and 90-day all-cause mortality as the primary outcome. Multiple logistic regression will be used to assess independent associations with mortality in addition to Receiver Operating Characteristics curves to identify cut-off Pi values associated with mortality and overcorrection. Linear mixed models will be conducted to assess Pi treatment effects. Subgroup analyses will be performed based on Pi abnormalities observed during ICU admission, categorized as normo-, hypo-, hyper-, or mixed, along with its severity (mild, moderate, or severe). DiscussionThe GUTPHOS study will be the first multicentre, prospective observational cohort study to investigate the prevalence, management practices, and consequent outcomes associated with Pi abnormalities during the first week of ICU admission. Its results may bridge the current evidence gap in repletion protocols while establishing the groundwork for a subsequent randomized controlled trial. Clinical trial registryNCT05909722.

Mechanisms of vacuolar phosphate efflux supporting soybean root hair growth in response to phosphate deficiency.

Phosphorus is an essential macronutrient for plant growth and development. In response to phosphate (Pi) deficiency, plants rapidly produce a substitutive amount of root hairs; however, the mechanisms underlying Pi supply for root hair growth remain unclear. Here, we observed that soybean (Glycine max) plants maintain a consistent level of Pi within root hairs even under external Pi deficiency. We therefore investigated the role of vacuole-stored Pi, a major Pi reservoir in plant cells, in supporting root hair growth under Pi-deficient conditions. Our findings indicated that two vacuolar Pi efflux (VPE) transporters, GmVPE1 and GmVPE2, remobilize vacuolar stored Pi to sustain cytosolic Pi content in root hair cells. Genetic analysis showed that double mutants of GmVPE1 and GmVPE2 exhibited reduced root hair growth under low Pi conditions. Moreover, GmVPE1 and GmVPE2 were highly expressed in root hairs, with their expression levels significantly upregulated by low Pi treatment. Further analysis revealed that GmRSL2 (ROOT HAIR DEFECTIVE 6-like 2), a transcription factor involved in root hair morphogenesis, directly binds to the promoter regions of GmVPE1 and GmVPE2, and promotes their expressions under low Pi conditions. Additionally, mutants lacking both GmRSL2 and its homolog GmRSL3 exhibited impaired root hair growth under low Pi stress, which was rescued by overexpressing either GmVPE1 or GmVPE2. Taken together, our study has identified a module comprising vacuolar Pi exporters and transcription factors responsible for remobilizing vacuolar Pi to support root hair growth in response to Pi deficiency in soybean.

Protective effects of Passiflora Incarnata on ischemia-reperfusion injury in testicular torsion: an experimental study in a rat model.

The current study aimed to explore the potential protective effect of Passiflora Incarnata L., (PI) in treating IR injury after testicular torsion in rats. This research investigated the impact of PI on IR damage in male Wistar albino rats. Animals were divided to three groups: group 1 (sham), group 2 (IR), and group 3 (IR+PI). The malondialdehyde (MDA), myeloperoxidase (MPO) and glutathione (GSH) levels did not significantly differ across the groups (p = 0.830, p = 0.153 and p=0.140, respectively). However, Group 3 demonstrated a superior total antioxidant status (TAS) value compared to Group 2 (p = 0.020). Concurrently, Group 3 presented a significantly diminished mean total oxidant status (TOS) relative to Group 2 (p = 0.009). Furthermore, Group 3 showed a markedly improved Johnsen score relative to Group 2 (p < 0.01). IR caused cell degeneration, apoptosis, and fibrosis in testicular tissues. PI treatment, however, mitigated these effects, preserved seminiferous tubule integrity and promoted regular spermatogenesis. Furthermore, it reduced expression of tumor necrosis factor-alpha (TNF-α), Bax, and Annexin V, signifying diminished inflammation and apoptosis, thereby supporting cell survival (p < 0.01, p < 0.01, p < 0.01, respectively). This study revealed that PI significantly reduces oxidative stress and testicular damage, potentially benefiting therapies for IR injuries.

Strigolactones Might Regulate Ovule Development after Fertilization in Xanthoceras sorbifolium.

Strigolactones (SLs) were recently defined as a novel class of plant hormones that act as key regulators of diverse developmental processes and environmental responses. Much research has focused on SL biosynthesis and signaling in roots and shoots, but little is known about whether SLs are produced in early developing seeds and about their roles in ovule development after fertilization. This study revealed that the fertilized ovules and early developing pericarp in Xanthoceras sorbifolium produced minute amounts of two strigolactones: 5-deoxystrigol and strigol. Their content decreased in the plants with the addition of exogenous phosphate (Pi) compared to those without the Pi treatment. The exogenous application of an SL analog (GR24) and a specific inhibitor of SL biosynthesis (TIS108) affected early seed development and fruit set. In the Xanthoceras genome, we identified 69 potential homologs of genes involved in SL biological synthesis and signaling. Using RNA-seq to characterize the expression of these genes in the fertilized ovules, 37 genes were found to express differently in the fertilized ovules that were aborting compared to the normally developing ovules. A transcriptome analysis also revealed that in normally developing ovules after fertilization, 12 potential invertase genes were actively expressed. Hexoses (glucose and fructose) accumulated at high concentrations in normally developing ovules during syncytial endosperm development. In contrast, a low ratio of hexose and sucrose levels was detected in aborting ovules with a high strigolactone content. XsD14 virus-induced gene silencing (VIGS) increased the hexose content in fertilized ovules and induced the proliferation of endosperm free nuclei, thereby promoting early seed development and fruit set. We propose that the crosstalk between sugar and strigolactone signals may be an important part of a system that accurately regulates the abortion of ovules after fertilization. This study is useful for understanding the mechanisms underlying ovule abortion, which will serve as a guide for genetic or chemical approaches to promote seed yield in Xanthoceras.

Dimensional Regulation from 1D/3D to 2D/3D of Perovskite Interfaces for Stable Inverted Perovskite Solar Cells.

Constructing low-dimensional/three-dimensional (LD/3D) perovskite solar cells can improve efficiency and stability. However, the design and selection of LD perovskite capping materials are incredibly scarce for inverted perovskite solar cells (PSCs) because LD perovskite capping layers often favor hole extraction and impede electron extraction. Here, we develop a facile and effective strategy to modify the perovskite surface by passivating the surface defects and modulating surface electrical properties by incorporating morpholine hydriodide (MORI) and thiomorpholine hydriodide (SMORI) on the perovskite surface. Compared with the PI treatment that we previously developed, the one-dimensional (1D) perovskite capping layer derived from PI is transformed into a two-dimensional (2D) perovskite capping layer (with MORI or SMORI), achieving dimension regulation. It is shown that the 2D SMORI perovskite capping layer induces more robust surface passivation and stronger n-N homotype 2D/3D heterojunctions, achieving a p-i-n inverted solar cell with an efficiency of 24.55%, which retains 87.6% of its initial efficiency after 1500 h of operation at the maximum power point (MPP). Furthermore, 5 × 5 cm2 perovskite mini-modules are presented, achieving an active-area efficiency of 22.28%. In addition, the quantum well structure in the 2D perovskite capping layer increases the moisture resistance, suppresses ion migration, and improves PSCs' structural and environmental stability.

Mineral and Carbon Metabolic Adjustments in Nodules of Symbiotically Grown Faba Bean (Vicia faba L.) Varieties in Response to Organic Phosphorus Supplementation.

Phosphorus (P) is a major limiting factor for legume and symbiotic nitrogen fixation (SNF). Although overall adaptations of legumes to P supplementation have been extensively studied in connection with inorganic P, little information is currently available regarding nodulation or SNF responses to organic P (Po) in hydroponics. We investigated the mineral and carbon metabolism of Po-induced nodules of two contrasting faba bean varieties grown hydroponically under inorganic P (Pi), viz., in P-deficient (2 µM KH2PO4, -Pi), sufficient-P (200 µM KH2PO4, +Pi), and phytic acid (200 µM, Po) conditions, and were inoculated with Rhizobium leguminosarum bv. viciae 3841 and grown for 30 days. The results consistently reveal similar growth and biomass partitioning patterns between +Pi and Po, with both varying substantially from -Pi. In comparison, +Pi and Po observed equivalent accumulations of overall elemental P concentrations, with both increasing by 114 and 119%, respectively, relative to -Pi. A principal component analysis on metabolites showed a clear separation of the -Pi treatment from the others, with +Pi and Po correlating closely together, highlighting the nonsignificant differences between them. Additionally, the δ15N abundance of shoots, roots, and nodules was not significantly different between treatments and varieties and exhibited negative δ15N signatures for all tissues. Our study provides a novel perspective on mineral and carbon metabolism and their regulation of the growth, functioning, and reprogramming of nodules upon phytate supply.

Intracytoplasmatic BCMA Point Mutation and Teclistamab Resistance: Genomic Assessment of a Multidrug-Resistant Multiple Myeloma Patient

Introduction Proteasome inhibitors (PIs) are standard drugs for the treatment of Multiple Myeloma (MM), bispecific antibodies (BsAbs) and CART cells are rapidly and successfully being integrated into treatment regimens. Still, several current studies have shown that drug resistance remains one of the biggest clinical challenges. For instance, deletions in BCMA (alias TNFRSF17) were shown to confer a target loss and therefore resistance to BCMA CARTs (Da Via et al. Nat Med 2021, Samur et al. Nat Commun 2021) as well as to a CD3xBCMA bispecific T cell-engager (Truger et al. Blood Adv 2021). Besides the extracellular ligand-binding domain, BCMA further consist of a membrane spanning and a cytoplasmatic region that activates cell functions. To date not much is known in regard to alterations affecting the latter and their role in resistance. Here we present genetic profiling of a multi-refractory MM patient who relapsed to PIs, anti- BCMA (Teclistamab) and anti- GPRC5D (Talquetamab) BsAbs. Methods We performed FISH and a targeted capture-hybridization DNA sequencing panel (tchDNA-Seq) as previously described in Rosa-Rosa et al. Cancers 2022 in two sequential samples: first a bone marrow biopsy at diagnosis and second, 43 months later, a FFPE biopsy of the retroperitoneal mass of a progression plasmacytoma, after PI but prior Teclistamab treatment. The MM-specific tchDNA-Seq panel includes over 100 genes involved in MM progression, drug resistance, immunotherapy and canonical loci to cover IG rearrangements and frequent translocations. In order to predict the impact of the observed gene mutations on the protein structure, we generated 3D projections using the PyMOL molecular visualization system and references from the Protein Data Bank. The KinasePhos3 program was used to predict the most probable kinase to phosphorylate new, by the mutation generated, sites. Results Via FISH, the patient was stratified as a high-risk patient as 13% of the tumor cells showed a del17p at diagnosis, 97% a t(4;14) translocation, and 88% chromosomal gains for 1q21 and 11q13. The patient initially responded well to VRd+HCT. After 3.6 years he relapsed and developed an abdominal plasmacytoma. He was non-responsive to the following Isa-Kd treatment and relapsed again, two months after Teclistamab induction. To the subsequent PomCyDex and Talquetamab treatments he was primary refractory. In the plasmacytoma, four SNVs were detected: PSMC5 p.G152V with a VAF of 42%, BCMA p.C128Y (3%) and the two B2M mutations B2M p.S14F (47%) and B2M p.L107F (11%). At diagnosis, the VAF was 1% for PSMC5 p.G152V and B2M p.S14F, the other two SNVs were absent. In B2M, mutations in residues 13-16 have been associated with impaired protein expression and complex formation (Challa-Malladi et al. Cancer Cell 2011). The PSMC5 SNV was topologically identified within a glycine-rich loop in the ATP binding pocket of the proteasome in direct interaction with the ATP amine group, therefore likely impacting the proteolytic capacity increasing the PI tolerance. B CMA p.C128Y is located in the C-terminal cytoplasmatic tail within a highly conserved 25-aa protein segment (position 119 to 143) that has been shown to be necessary for TRAF association and NF-κB, Elk-1, and JNK activation (Hatzoglou et al. J Immunol 2000). A cysteine is mutated to a tyrosine. With high probability, this new site will be phosphorylated by kinases of the c-Src family (score 0.92), e.g. Fyn (0.71) or Lyn (0.66). Moreover, this new phosphotyrosine would become a binding site for the kinase SH2 domain (Diop et al. Int J Mol Sci 2022). Conclusions Here we report emerging mutations in PSMC5 and BCMA, that are likely causal for PI and Teclistamab resistance. PSMC5 p.G152 represents a mutational hotspot as four more PI-resistant patients with substitutions were recently reported in Haertle et al. Cancers 2023. The intracytoplasmatic BCMA SNV creates a new kinase phosphorylation and binding site resulting in aberrant downstream signaling. B2M forms part of the MHC1 molecules, responsible for antigen presentation to CD8+ T cells and the B2M serum level is a prognostic indicator in MM. However, the role of B2M alterations in disease progression and drug resistance needs to be elucidated. Predicting the biological impact based on 3D structural models remains speculative, thus, functional confirmation introducing the BCMA and B2M alterations in cell line models by genetic engineering is pending.

THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells

Abstract Disclosure: K.A. Alghamdi: None. D. Ward: None. Calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and we reported recently that pathophysiologic phosphate (Pi) concentrations can attenuate CaR activity directly in transfected HEK-293 cells and can increase PTH secretion from human and murine parathyroid cells (1). This was investigated further using thyroidal C cell-derived TT cells (ATCC), which express CaR endogenously. Intracellular Ca2+ (Ca2+i) mobilisation was assayed by epifluorescence microscopy, calcitonin secretion by ELISA, protein expression by immunoblotting and CaR mRNA expression by qRT-PCR. Co-stimulation of TT cells with the (CaR-activating) calcimimetic R568 (1µM) and spermine (1mM) elicited robust Ca2+i mobilisation, that was inhibited (33±4%; P&amp;lt;0.001; N=4) by 2mM (pathophysiologic) Pi-containing buffer vs 0.8mM Pi control. In contrast, raising Pi concentration was without effect on carbachol-induced Ca2+i mobilisation (acting via muscarinic receptors) (P=0.89; N=3). Also, 1.2mM (high) sulphate elicited a similar CaR inhibition as for Pi vs 0.3mM control (-28±16%; P&amp;lt;0.05; N=4). Next, it was found that CaR-mediated stimulation of the TT cells with 1µM R568 &amp; 1mM spermine (in 2mM Ca2+ and 0.8mM Pi) increased calcitonin secretion, as expected, whereas the same stimulation in 2mM Pi (in the presence of 2.2mM Ca2+ to correct for any reduction in free Ca2+) significantly blunted this response (-59.7±15%; P&amp;lt;0.05;N=4). Interestingly, CaR mRNA expression was significantly downregulated 45±13%; P&amp;lt;0.05;N=6) after 48 hours treatment with media containing 2mM Pi (vs 0.8mM control). However, while CaR protein expression in the TT cells was confirmed by immunoblotting, its abundance was unaffected by the same 2mM Pi cotreatment (P=0.79; N=8). Therefore, pathophysiologic Pi treatment inhibits endogenous CaR-induced signalling, calcitonin secretion and CaR mRNA expression in thyroidal TT cells but without affecting CaR abundance. These results further support the idea that the CaR represents a mineral sensor, at which Pi acts directly as a non-competitive antagonist to limit CaR-induced suppression of PTH secretion. Reference: (1) Centeno et al. Nat Commun. 2019;10:4693. Presentation: Thursday, June 15, 2023

#3986 THE ROLE AND MECHANISM OF EZH2 IN VASCULAR CALCIFICATION ASSOCIATED WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Vascular calcification (VC) is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. EZH2 (Enhancer of zeste 2 homolog-2) is reported as a key epigenetic regulator involved in various kidney diseases such as acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. In this study, we aimed to investigated the role of EZH2 in chronic kidney disease associated vascular calcification. Method 1. Patients and Radial Artery Analysis Radial arteries with or without calcification from patients with end-stage renal disease that underwent arterial venous fistular surgery were collected for Von Kossa staining and immunohistochemical (IHC) staining. 2. Animal model of Chronic renal failure-Vascular Calcification (CRF-VC) (in vivo) An adenine and phosphate (1.2%) diet-induced CRF mouse model was designed following an 8-week program. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition and histological analysis. Plasma levels of Alkaline phosphate (ALP), and phosphate (Pi) were measured. The transcription level of EZH2 and Runt-related transcription factor 2 (Runx2) were measured by real-time PCR and IHC staining. 3. Aortic ring calcification (ex vivo) Thoracic aortas were dissected from SD rat. To induce calcification, Pi (NaH2PO4/Na2HPO4 [pH = 7.4]) was added to HG-DMEM at a final concentration of 2.6 mmol/l. After the indicated incubation periods (3, 5, 7 days), samples were taken for Calcium deposition assay, Western blotting and histological analysis. 4. Cell culture and treatment (in vitro) Calcification of rat smooth muscle embryonic thoracic aorta cell line A7r5 (VSMC) was induced by incubation in calcifying media. 3-deazaneplanocin A (3-DZNeP), a carbocyclic analog of adenosine was added right after Pi to block EZH2 activation. After the indicated incubation periods, samples were collected for Calcium deposition assay and Western blotting analysis. Results 1. In vivo, EZH2 protein level was increased in calcified radial arteries from patients with end-stage renal disease that underwent arterial venous fistular operation. In CRF-VC mice model, the aortic calcium deposition markedly aggravated in the group induced by adenine diet. Severe vascular calcification was also induced as shown by increased intensity of von Kossa. Moreover, IHC and q-PCR results showed that the protein and transcriptional levels of EZH2 expression were upregulated in calcified aortas of CRF mice, in paralleled with the increased expression Runx2 in protein and transcriptional levels. 2. Ex vivo, histological analysis revealed significant medial vascular calcification in the aortic ring culture with this calcifying medium for 7 days. Similarly, WB results showed that the expression of EZH2 and its downstream, methylation of Histone H3 at lysine 27 (H3K27me3), were significantly enhanced after high Pi medium stimulation while the osteogenic markers Runx2, Osteopontin (OPN) and Msh homeobox 2 (Msx2) protein levels were upregulated and the expression of VSMC differentiation markers α-Smooth muscle actin (α-SMA), Calponin (CNN) and Smooth muscle protein 22 (SM22) were downregulated. 3. In vitro, the osteogenic markers Runx2, OPN and Msx2 were significantly upregulated in high Pi treated VSMCs compared to control VSMCs. Moreover, the epigenetic markers EZH2 and H3K27me3a were markedly repressed in calcifying VSMCs by time. 4. 3-DZNep treatment reduced the calcium deposition in a concentration dependent manner. Western blotting showed that 3-DZNep treatment blocked the increased expression of osteogenic markers Runx2, Bone morphogenetic protein 2 (BMP2) as well as epigenetic markers EZH2 and H3K27me3a by high Pi treatment, indicating that inhibition of EZH2 attenuates VSMCs Calcification. Conclusion Our study revealed that EZH2 is highly expressed in calcified vascular tissues of CRF patients and CRF mice, rat aorta culture and VSMCs calcification models, and EZH2 inhibitor 3-DZNep attenuated calcification of VSMCs. EZH2 could be a promising target for treatment of vascular calcification in CRF patients.

Analysis of the prevalence and risk factors of pressure injuries in the hospitalized pediatric population: A retrospective study

BackgroundPressure injury (PI) is an essential indicator of the quality of nursing care and affects hospitalized newborns and children. However, studies on the prevalence of PI and associated risk factors in children are limited. AimsThis study aimed to analyze the prevalence of PI and risk factors affecting the development of PI in the hospitalized pediatric population. MethodsThis was a descriptive, retrospective study. Data were obtained via electronic medical records of 6350 pediatric patients admitted to a university hospital between January 2019 and April 2022. Ethics committee approval was obtained. Patient medical records and data associated with PI and medical treatment were collected through the ‘Information Form,’ ‘Braden Scale,’ ‘Braden Q Scale,’ ‘Pressure Ulcer Staging Form,’ and ‘Pediatric Nutrition Risk Score (PNRS).’ Data were analyzed using descriptive statistics, correlation analysis, Mann-Whitney U test, Kruskal Wallis test, and Multilinear Regression analysis. ResultsMore patients (66.2%) were males, and 49.2% of the children were 0–12 months old. 2368 out of 6350 pediatric patients were treated in the PICU. It was determined that a total of 143 PI occurred in 59 patients from PICU. The PI prevalence was 2.25% for all patients and 6.04% for PICU patients. Twenty-one percent of the patients had medical device-related PI (MDRPIs), 35.7% of PI occurred in the occiput, 13.3% in the coccyx/sacrum, and 67.1% of PI was Deep Tissue Injury. In the multiple regression model, children's albumin level, hemoglobin level, PNRS scores, Body Mass Index, and length of hospital stay significantly affected BRADEN scores. They were explained 30.3% of their scores of Braden. ConclusionDespite the limitations of the retrospective study, the prevalence of PI in the pediatric population in this study was lower than that reported in previous studies, but the prevalence of MDRPIs was higher. Based on the study results, it is recommended to implement preventive interventions for MDRPIs and plan prospective studies.

Mechanisms of preferential bone formation in myeloma bone lesions by proteasome inhibitors

Proteasome inhibitors (PIs) can preferentially restore bone in bone-defective lesions of patients with multiple myeloma (MM) who respond favorably to these drugs. Most prior in vitro studies on PIs used continuous exposure to low PI concentrations, although pharmacokinetic analysis in patients has shown that serum concentrations of PIs change in a pulsatile manner. In the present study, we explored the effects of pulsatile treatment with PIs on bone metabolism to simulate in vivo PI pharmacokinetics. Pulsatile treatment with bortezomib, carfilzomib, or ixazomib induced MM cell death but only marginally affected the viability of osteoclasts (OCs) with F-actin ring formation. Pulsatile PI treatment suppressed osteoclastogenesis in OC precursors and bone resorption by mature OCs. OCs robustly enhanced osteoblastogenesis in cocultures with OCs and MC3T3-E1 pre-osteoblastic cells, indicating OC-mediated coupling to osteoblastogenesis. Importantly, pulsatile PI treatment did not impair robust OC-mediated osteoblastogenesis. These results suggest that PIs might sufficiently reduce MM cell-derived osteoblastogenesis inhibitors to permit OC-driven bone formation coupling while suppressing OC differentiation and activity in good responders to PIs. OC-mediated coupling to osteoblastogenesis appears to be a predominant mechanism for preferential occurrence of bone regeneration at sites of osteoclastic bone destruction in good responders.

Cryopreserved platelets and amotosalen-treated plasma in an experimental clot formation set-up.

Amotosalen treatment of plasma and cryopreservation of platelets affect the quality and potentially the interplay between platelets and coagulation factors. We set up an experimental clot formation study to test the hypothesis that amotosalen treatment of plasma affects the interaction with different platelet preparations. Pooled plasma units (n=16) were subjected to coagulation tests before and after pathogen inactivation with amotosalen treatment (PI) and aliquots were frozen at -80°C. Fresh and cryopreserved platelets were analyzed for phenotypic and activity markers. Finally, coagulation properties of different combinations of platelets and plasma, before and after PI, were analyzed by viscoelastography (ROTEM). PI of plasma reduced the concentration of several coagulation factors (p<0.01). Cryopreservation altered phenotypic expression and reduced the platelets' ability to respond to agonists (p<0.0001). The interplay between all plasma derivatives and cryopreserved platelets resulted in shortened coagulation time (p<0.0001) but prolonged clot formation time and reduced clot strength (p<0.0001) as compared to the interaction between fresh platelets with different plasma variants. PI of the plasma does not seem to have a major impact on coagulation time, clot formation time or clot strength. Our data show that the reduced concentration of coagulation factors after PI treatment of plasma are negligible measured by viscoelastography, with fresh and cryopreserved platelets in this experimental clot formation setup, and that platelets play a more pronounced role. Cryopreserved platelets are more activated and result in reduced clot stability.

Allochthonous arbuscular mycorrhizal fungi promote Salix viminalis L.–mediated phytoremediation of polycyclic aromatic hydrocarbons characterized by increasing the release of organic acids and enzymes in soils

Polycyclic aromatic hydrocarbons (PAHs) are well known persistent organic pollutants that have carcinogenic, teratogenic, and mutagenic effects on humans and animals. Arbuscular mycorrhizal fungi (AMF) that can infest plant hosts and form symbioses may help plants to enhance potential rhizosphere effects, thus contributing to the rhizodegradation of PAH-contaminated soils. The present study aimed to assess the effectiveness of AMF on enhancing Salix viminalis–mediated phytoremediation of PAH-polluted soil and clarify the plant enzymatic and organic acid mechanisms induced by AMF. Natural attenuation (NA), phytoremediation (P, Salix viminalis), S. viminalis-AMF combined remediation using willow inoculated with Funneliformis mosseae (PM), Laroideoglomus etunicatum (PE), and Rhizophagus intraradices (PI) were used as strategies for the remediation of PAH-polluted soils. The results showed that AMF inoculation contributed to the dissipation of the high-molecular-weight PAH benzo (α) pyrene that had concentrations in PM, PE, and PI treatments of 40.1 %, 24.49 %, and 36.28 % of the level in the NA treatment, and 62.32 %, 38.05 %, and 56.38 % of the level in the P treatment after 90 days. The mycorrhizal treatment also improved the removal efficiency of phenanthrene and pyrene, as their concentrations were sharply decreased after 30 days compared to the NA and P treatments. The research further clarified the changes in rhizosphere substances induced by AMF. Organic acids including arachidonic acid, octadecanedioic acid, α-linolenic acid, 10,12,14-octadecarachidonic acid and 5-methoxysalicylic acid that can act as co-metabolic substrates for certain microbial species to metabolize PAHs were significantly increased in AMF-inoculated treatments. AMF inoculation also elevated the levels of polyphenol oxidase, laccase, and dehydrogenase, that played crucial roles in PAHs biodegradation. These findings provide an effective strategy for using AMF-assisted S. viminalis to remediate PAH-polluted soils, and the results have confirmed the key roles of organic acids and soil enzymes in plant-AMF combined remediation of PAHs.

Transcriptome Level Analysis of Genes of Exogenous Ethylene Applied under Phosphorus Stress in Chinese Fir.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a widely grown gymnosperm in China. Phosphorus (P) is an indispensable nutrient for the growth of Chinese fir. Inorganic phosphate (Pi) deficiency exists in soils of many Chinese fir planting area regions, and the trees themselves have limited efficiency in utilizing P from the soil. Ethylene is important in regulation responses to nutrient deficiencies. However, little is known about how ethylene signals participate in Pi stress in Chinese fir. A total of six different treatments were performed to reveal the transcript levels of Chinese fir under Pi, ethephon (an ethylene-releasing compound), and CoCl2 (cobalt chloride, an ethylene biosynthesis inhibitor) treatments. We assembled a full-length reference transcriptome containing 22,243 unigenes as a reference for UMI RNA-seq (Digital RNA-seq). There were 586 Differentially Expressed Genes (DEGs) in the Pi starvation (NP) group, while DEGs from additional ethephon or CoCl2 in NP were 708 and 292, respectively. Among the DEGs in each treatment, there were 83 TFs in these treatment groups. MYB (v-myb avian myeloblastosis viral oncogene homolog) family was the most abundant transcription factors (TFs). Three ERF (Ethylene response factor) family genes were identified when only ethylene content was imposed as a variable. Enrichment analysis indicated that the ascorbate and aldarate metabolism pathway plays a key role in resistance to Pi deficiency. This study provides insights for further elucidating the regulatory mechanism of Pi deficiency in Chinese fir.

Integration of small RNA, degradome, and transcriptome sequencing data illustrates the mechanism of low phosphorus adaptation in Camellia oleifera.

Phosphorus (P) is an indispensable macronutrient for plant growth and development, and it is involved in various cellular biological activities in plants. Camellia oleifera is a unique high-quality woody oil plant that grows in the hills and mountains of southern China. However, the available P content is deficient in southern woodland soil. Until now, few studies focused on the regulatory functions of microRNAs (miRNAs) and their target genes under low inorganic phosphate (Pi) stress. In this study, we integrated small RNA, degradome, and transcriptome sequencing data to investigate the mechanism of low Pi adaptation in C. oleifera. We identified 40,689 unigenes and 386 miRNAs by the deep sequencing technology and divided the miRNAs into four different groups. We found 32 miRNAs which were differentially expressed under low Pi treatment. A total of 414 target genes of 108 miRNAs were verified by degradome sequencing. Gene ontology (GO) functional analysis of target genes found that they were related to the signal response to the stimulus and transporter activity, indicating that they may respond to low Pi stress. The integrated analysis revealed that 31 miRNA–target pairs had negatively correlated expression patterns. A co-expression regulatory network was established based on the profiles of differentially expressed genes. In total, three hub genes (ARF22, WRKY53, and SCL6), which were the targets of differentially expressed miRNAs, were discovered. Our results showed that integrated analyses of the small RNA, degradome, and transcriptome sequencing data provided a valuable basis for investigating low Pi in C. oleifera and offer new perspectives on the mechanism of low Pi tolerance in woody oil plants.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale

BackgroundOrchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination.ResultsThe two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation.ConclusionsCompatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Genome-Wide Analysis of DoSPX Genes and the Function of DoSPX4 in Low Phosphorus Response in Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a famous Chinese herb. D. officinale grows on rocks where the available phosphorus is low. The SPX family plays a critical role in maintaining Pi homeostasis in plants. In this paper, 9 SPX family genes were identified in the genome of D. officinale. Bioinformatics and qRT-PCR analysis showed that DoSPXs were involved in response to −Pi stress and had different expression patterns. DoSPX4, which had a unique expression pattern, was clustered with AtSPX4 and OsSPX4. Under −Pi treatment, the expression level of DoSPX4 reached a peak on 5 d in roots, while showing a downward trend in the aboveground parts. DoSPX4 was located on the cell membrane. Overexpression DoSPX4 promoted Pi content in the stem and the expression level of NtPHT1/2 in Nicotiana tabacum. The results of Yeast two-hybrid showed that DoSPX4 could interact with Phosphate High-Affinity Response factor (DoPHR2). These results highlight the role of DoSPX4 in response to low phosphorus, which provides a theoretical basis for further study on the response mechanism of −Pi in D. officinale.

S184: EVALUATING TECLISTAMAB IN PATIENTS WITH RELAPSED/ REFRACTORY MULTIPLE MYELOMA FOLLOWING EXPOSURE TO OTHER B-CELL MATURATION ANTIGEN (BCMA)-TARGETED AGENTS

Background: Teclistamab (JNJ-64007957) is a T cell redirecting bispecific antibody that targets both B-cell maturation antigen (BCMA) and CD3 receptors to induce T cell mediated cytotoxicity of BCMA-expressing myeloma cells. MajesTEC-1 is an open-label, multicohort, phase 1/2 study evaluating teclistamab in patients (pts) with relapsed/ refractory multiple myeloma (RRMM) previously treated with ≥3 prior lines of therapy (LOT). An overall response rate (ORR) of 62.0% in pts with no prior anti-BCMA treatment (tx) was previously reported in a pooled analysis from phase 1 and phase 2 cohort A at a median follow-up of 7.8 mo. Aims: We report efficacy and safety results of teclistamab from cohort C, which enrolled patients who had prior exposure to anti-BCMA treatment. Methods: Eligible pts (age ≥18 y) had multiple myeloma (MM) per IMWG criteria and were previously treated with ≥3 prior LOT, including a PI, IMiD, anti-CD38 antibody, and anti-BCMA treatment (chimeric antigen receptor T cell therapy [CAR-T] or Ab drug conjugate [ADC]). Pts were enrolled using a Simon’s stage design to receive weekly subcutaneous teclistamab 1.5 mg/kg (step-up doses of 0.06 and 0.3 mg/kg). ORR per IMWG 2016 criteria was the primary endpoint. All AEs were graded per CTCAE v4.03; immune effector cell–associated neurotoxicity syndrome (ICANS) and cytokine release syndrome (CRS) were graded per ASTCT guidelines. Results: In cohort C, 38 pts (median age 63.5 y [range 32–82]; 63% male) received teclistamab (median prior LOT 6 [range 3–14]) at the data cutoff date of Sep 7, 2021. Of the 38 patients, 25 (66%) were refractory to an anti-BCMA treatment, and 32 (84%) were refractory to last LOT. Among 25 efficacy-evaluable patients, 16 (64%) received prior ADC, 11 (44%) received prior CAR-T, and 2 pts received both. The ORR was 40% (95% CI, 21–61) at a median follow-up of 6.9 mo (range 0.7–8.7). Complete response or better were observed in 5 pts (20%). In ADC-exposed and CAR-T-exposed pts, the ORR was 38% (95% CI, 15–65) and 45% (95% CI, 17–77), respectively. Responses were rapid in most, with deepening of responses over time in 7 of 25 pts. While the median duration of response was not reached, median time to first response was 1.2 mo (range, 0.2–4.9) and to best response was 2.1 mo (range, 1.1–5.7). No new safety concerns were observed, and the safety profile was comparable with that of BMCA tx-naive pts. Infections were reported in 16 pts (42%; grade 3/4, 26%). Most common AEs (n=38) were CRS (63%; all grade 1/2; median time to CRS onset: 3 d [range, 2–6], duration of CRS: 2 d [range, 1–4]), thrombocytopenia (42%; grade 3/4, 29%), neutropenia (55%; grade 3/4, 50%), lymphopenia (40%; grade 3/4, 37%), and anemia (39%; grade 3/4 29%), Grade 3 ICANS was reported in 1 pt which resolved with supportive care and the pt remained on tx. Anti-teclistamab Abs were not detected in any pts. Baseline BCMA expression levels were comparable with those reported in BCMA tx-naive pts. Updated efficacy and safety results will be presented for 40 pts. Summary/Conclusion: These preliminary results observed with serial targeting of BCMA with teclistamab following ADC or CAR-T tx suggest a promising ORR with early responses that deepen over time. Additionally, a well-tolerated safety profile was observed in patients treated with anti-BCMA tx.