Renal diseases impose considerable health and economic burdens on health systems worldwide, and there is a lack of efficient methods for the prevention and treatment due to their complexity and heterogeneity. Kidneys are organs with a high demand for energy produced by mitochondria, in which Rrm2b has critical functions as reported. The Rrm2b kidney-specific knockout mice we generated exhibited age-dependent exacerbated features, including mitochondrial dysfunction and increased oxidative stress; additionally, resulted in severe disruption of mitochondria-related metabolism. Rrm2b is vital not only to supply dNTPs for DNA replication and repair, but also to maintain structural integrity and metabolic homeostasis in mitochondria. Thence, Rrm2b deletion might induce chronic kidney defects in mice. This model can facilitate exploration of novel mechanisms and targeted therapies in the kidney diseases and has important translational and clinical implications. Ribonucleotide reductase M2B (Rrm2b), also known as p53R2, is a subunit of the ribonucleotide reductase complex. Rrm2b is a p53-inducible gene that was first identified in cancer-derived human cells with a highly regulated p53 expression system3. Rrm2b plays critical roles in DNA repair, mitochondrial DNA (mtDNA) synthesis, oxidative stress resistance, cell cycle regulation and metastasis suppression8. Kimura and colleagues generated Rrm2b-deficient mice, which developed normally until weaning and then showed growth retardation and early mortality9. Severe renal failure, caused by activation of p53-dependent apoptosis, was found in Rrm2b-deficient mice. Thence, Rrm2b seems to have critical function in kidney, but its mechanism is unclear. Rrm2b can resist oxidative stress by scavenging reactive oxygen species (ROS) and is involved in the control of mitochondria homeostasis, including structural integrity and functional capacity13. In Rrm2b knockout mouse embryonic fibroblasts (MEFs), dNTP pools were severely attenuated under oxidative stress conditions. Rrm2b deficiency also causes severe mtDNA deletion in various tissues in mice4. Recent studies have unveiled that cooperation between RRM2B and ∆1-pyrroline-5-carboxylate reductases 1 and 2 (PYCRs) can directly or indirectly modulate antioxidative responses in human telomerase reverse transcriptase (hTERT)-immortalized normal human foreskin fibroblasts (HFFs-hTERT)14. Compromised DNA repair in the nucleus and mitochondria can result in profound DNA damage and affect mitochondrial homeostasis, which can subsequently increase oxidative stress and cellular damage. Rrm2b plays an important role in DNA repair and maintenance of mitochondrial functions. In this study, we generated a mouse model with conditional knockout of Rrm2b in the renal tubular epithelium to study the effects of Rrm2b deletion on mitochondria-related and renal functions. We propose that Rrm2b deletion damages mitochondrial integrity and impedes the mitochondrial functions, resulting in the disruption of the homeostasis of mitochondrial metabolisms.Ribonucleotide reductase  catalyzes the rate-limiting step in the 15, the Cdh-16  promoter was activated in the tubular epithelial cells of the kidney16. Ksp-cadherin was found to be expressed in the basolateral membrane of renal tubular epithelial cells and collecting duct cells, but not in glomeruli, blood vessels, or renal interstitial cells. After two generations of breeding with the Rrm2b F/F mice, kidney-specific Rrm2b KO mice were obtained and verified using regular PCR genotyping. The mice were bred and maintained in a specific pathogen-free facility. Euthanasia was performed using CO2 inhalation. The animal protocol was approved by the Institutional Animal Care and User Committee of National Defense Medical Center and all methods were performed in accordance with the relevant guidelines and regulations.An Rrm2b targeting vector containing two direct repeats of LoxP sites that flanked exon 3 to exon 5 of the Rrm2b gene was used as a set of homologous recombination arms to create an Rrm2b floxed allele . The targeted ES cell clones (C57BL/6 strain background) were screened by Southern blot analysis using designed probes. In Cdh16-Cre transgenic mice purchased from the Jackson Laboratory (JAX 012237)® Fast Universal PCR Master Mix and a real-time PCR instrument . Amplification was executed in triplicates for each RNA sample and primer set.Total RNA was isolated from the kidney cortex using TRIzol Reagent (Life Technology). We performed real-time quantitative PCR using a TaqMan probe with TaqManTissue samples were homogenized in lysis buffer with complete protease inhibitor and phosphatase inhibitor cocktails (Roche) and then denatured in SDS sample buffer in a boiling water bath. The total extracted proteins were separated on an SDS–polyacrylamide gel (Bio-Rad) and transferred to a Hybond N+ membrane . The membranes were blocked with 5% (w/v) nonfat dry milk, incubated with primary antibodies against Rrm2b  and Hsp70  and washed, and the proteins were detected using a Visualizer Kit (Millipore). Free glycerol in the serum and urine were measured calorimetrically using commercially available kits (Randox Lab Ltd.).17. The IHC staining was performed by incubating sections with primary antibodies, including antibodies targeting Rrm2b , 4-Hydroxynonenal (4-HNE) , and ki67 , for 18–24 h at 4 °C, and detection was performed using biotinylated secondary antibodies and an LSAB Kit (DakoCytomation). The specific staining for kidney sections was performed using a Gomori trichrome staining kit  and Picosirius red staining kit (Atom Scientific).Tissues were fixed in formalin buffered with phosphate and embedded in paraffin. Tissue sections (4 µm) were subjected to hematoxylin-eosin (H&E), immunohistochemistry (IHC) and special staining following standard proceduresMouse tissues were fixed in cacodylate buffer with glutaraldehyde (1.5%) and paraformaldehyde (1.5%), post-fixed in 1% OsO4 and rinsed in cacodylate buffer. Following dehydration, tissues were embedded in Epon and sectioned for TEM. The tissue ultrastructure was observed using a transmission electron microscope .18. Mitochondrial genomic DNA was co-purified with nuclear genomic DNA, and quantitative real-time PCR was performed for detection of mt-Co2  and 18S rRNA . The relative mtDNA copy number was calculated via normalization to 18S rRNA gene expression (mtDNA/nDNA)20.Oxygen consumption rate (OCR) of isolated mitochondria from tissues (kidney cortex) were measured using a Seahorse XFe Extracellular Flux Analyzer (Seahorse bioscience), and the results were normalized according to the protein contents of each sample. Mitochondria isolation from tissues was performed as previously described24. Briefly, the frozen tissue (50 ~ 100 mg) was homogenized on ice in a 500-µL mixture of chloroform, methanol and water  and centrifuged at 13,000 rpm for 10 min at 4 °C, and 150 µL of the supernatant was subsequently transferred to a sample vial. The remaining pellet was rehomogenized in 500 µL of methanol, followed by a second centrifugation. Another 150 µL aliquot of supernatant was added to the same sample vial, and then, the sample was dried. The dried extract was then derivatized with 80 µL of methoxyamine (15 mg/mL in pyridine) at 30 °C for 90 min. A 10-µL aliquot of retention index compound mixture  was added to the derivatized sample. The second step of the derivatization was performed by adding 80 µL of N-O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (1% trimethylchlorosilane) and incubating the sample at 70 °C for 120 min. The samples were subjected to gas chromatography-time of flight mass spectrometry (GC-TOF-MS) analysis directly after derivatization.Sample preparation and metabolite quantification were performed according to methods described previouslyMammalian metabolite standards were obtained from Sigma-Aldrich , Santa Cruz Biotech.  or Avanti polar lipids . The standards  were prepared in appropriate solutions and analyzed by GC-TOFMS to establish an in-house metabolite database.The separation analysis was performed using an Agilent 7890N gas chromatograph in splitless mode connected to a time-of-flight mass spectrometer . Separation was achieved using an Rxi-5ms capillary column , with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The GC oven temperature program was as follows: 2 min at 80 °C, raised by 10 °C/min to 140 °C, by 4 °C/min to 180 °C, by 10 °C/min to 240 °C and by 25 °C/min to 290 °C, followed by hold for 4.5 min at 290 °C. The temperatures of the injection, transfer interface, and ion source were set to 270, 270, and 220 °C, respectively. Mass spectra were obtained with electron impact ionization (70 eV) at full scan mode (m/z 30–600) and an acquisition rate of 25 spectra/s.25. Internal standards and any known artificial peaks, such as peaks caused by noise, column bleed and the BSTFA derivatization procedure, were removed from the data set. Metabolites were annotated through comparison with the reference standards in our in-house library. Commercially available mass spectral databases, such as the NIST library 2010 and the LECO/Fiehn Metabolomics Library, were also used for additional compound annotation (with a similarity threshed of 70%). The mass spectral data were subjected to log2 transformation and normalized by autoscaling.Data acquisition was performed using ChromaTOF software . MATLAB  software was used to perform data pretreatment procedures, such as baseline correction, de-noising, smoothing, alignment, time-window splitting, and multivariate curve resolutionAll results are presented as the mean ± SD of at least three independent samples. Comparisons between two groups were calculated using Student’s t test. Statistical differences between two groups were analyzed, and the differences were considered significant when p < 0.05.flx/flx; Cdh16-Cre/+, abbreviated to Rrm2b kiKO)  mice at 3 and 6 months of age  and a slight increase in the ketone concentration in 6-month-old kiKO mice compared with that in Rrm2b F/F mice.  and apurinic/apyrimidinic endonuclease 1 (Apex1/Ref1)  and superoxide dismutase 1 (Sod1)  was also observed  and proliferating cell nuclear antigen (Pcna)  Fig. . WesternKO) Fig. . Rrm2b kKO) Fig. , withoutKO) Fig. . Serum be. Table . Pale ane. Table . Tubule e. Table , S2a,b. e. Table . A margie. Table . Additioe. Table . The defd1) Fig. . Interesved Fig. . We alsona) Fig. , in Rrm2The mitochondria in the proximal renal tubular cells of the wild-type kidneys showed typical basal striping characteristics Fig. . In Rrm2To systematically understand the effects of Rrm2b-deletion in the kidney, we performed metabolic profiling analysis of the kidneys of Rrm2b kiKO and F/F mice. GC-TOF-MS analysis identified a total of 126 metabolites; of these, 13 metabolites exhibited lower concentrations and 14 exhibited higher concentrations in Rrm2b kiKO mice than in control mice Table . In the in situ, in situ reporter (knock-in), IHC, western blot, RT-PCR and/or northern blot analysis. The prediction scores of MitoProt, TargetP, MitoFates and MitoMiner were used as evidence of mitochondrial localization. Of the 342 genes, 67 encoded candidate mitochondrial proteins , betaine-homocysteine methyltransferase 2 (Bhmt2), dimethylglycine dehydrogenase precursor (Dmgdh) and glycerol kinase (Gk) responsible for glycolysis, glycerolipid and amino acid metabolism were repressed. Interestingly, an increase in Bhmt expression caused the high levels of dimethylglycine (DMG) that were observed under Rrm2b deficiency conditions ; aldehyde dehydrogenase 18 family, member A1 ; and ornithine transcarbamylase (Otc), were elevated  and identified 342 genes that are annotated as expressed in the renal cortex, as confirmed by RNA ons Fig. . In addited Fig. . There wted Fig. . Finallyted Fig. . All of 26. Additionally, Rrm2b has the capacity to scavenge ROS to protect against oxidative stress10. Rrm2b-deficient human and mouse models exhibit detectable decreases in the dNTP pool and DNA repair efficiency, resulting in severe damage to nuclear and mitochondrial DNA9. In addition, loss of Rrm2b led to declines in ROS scavenging and antioxidant capacity, damaged mitochondrial structures and exacerbated impairment of mitochondrial functions. All of these effects accelerated cellular oxidative stress in organisms  is found in the cytosol, nucleus, and mitochondria and is highly expressed in the kidney, liver and brain. Biochemically, Aldh7a1 catalyzes the conversion of D-glyceraldehyde to D-glycerate, which can enter the glycolysis pathway. Aldh7a1 can also metabolize betaine aldehyde to betaine to counteract hyperosmotic stress29. Additionally, Aldh7a1 can attenuate osmotic stress-induced apoptosis. However, Rrm2b deletion led to reduced Aldh7a1 expression and induced extreme oxidative stress and osmotic stress. Bhmt and Dmgdh are expressed in the proximal tubules of the renal cortex31. Aberrant expression of these two proteins under Rrm2b deficiency conditions caused DMG accumulation and decreases in L-homocysteine (Hcy) and serine levels, which consequently disrupted amino acid metabolism , while the proline oxidase (Pox) that converts proline to P5C showed no obvious difference in expression. The up-regulation of proline biosynthesis genes can trigger the conversion of glutamine towards proline and oxidizing NAD(P)H to NAD(P) + molecules. The activation of Pycr1 and P5cs resulted from Rrm2b deletion maybe a compensatory mechanism that cells used to counteract the increasing production of ROS.Increased Pycr and P5cs expression levels facilitate proline generation and indirectly enhance ROS generation while reducing TCA cycle fluxBased on our study, Rrm2b deletion in renal tubular cells caused several pathological defects in mice. Additionally, these defects were exacerbated with age, and some defects were similar to those observed in human disorders, as previously reported.34. Human patients with this disease have a bicarbonate reabsorption defect in the kidney and intestine. Rrm2b deficiency in mice caused metabolic defects, functional loss of renal tubules, and maybe impaired bicarbonate reabsorption from urine in proximal tubular cells.The proximal tubules regulate acid-base balance by secreting ammonia and reabsorbing filtrated bicarbonate. Urine biochemical analysis showed lower pH values in 6-month-old Rrm2b kiKO mice but not in younger mice; accordingly, we hypothesize that Rrm2b deletion may induce renal tubular acidemia type 2, also known as proximal renal tubular acidemia35. Although the effects of Rrm2b deficiency in the loop of Henle and collecting tubes are mostly unknown, oxidative damage mediated by mitochondria may lead to pathological changes in the loop of Henle and collecting tubes. Past studies have demonstrated that enhanced Rrm2b expression can reduce oxidative stress in both normal and oxidative-damage conditions12. Our data showed that Rrm2b deletion in renal tubular cells caused an increase in oxidative stress and a decrease in antioxidant capacity, which led to both morphological and functional mitochondrial defects. Additionally, exposure to oxidative stress can trigger increased expression of Pycr137, and indeed, we found enhanced expression of Pycr1 and Nrf2, another oxidative stress-induced gene, in our Rrm2b kiKO model. This suggests that Rrm2b deletion caused oxidative damage in the kidney. One of the critical functions of Pycr1 is to maintain mitochondrial function and integrity under oxidative damage conditions37. However, the compensatory effects of Pycr1 and other stress-induced genes were not sufficient to counteract and protect mitochondria against oxidative damage under Rrm2b deletion conditions. Furthermore, impaired antioxidant mechanisms and reduced ROS clearance can lead to oxidative damage and mitochondrial breakdown.The kidney is highly susceptible to ROS-induced damage. In addition, renal proximal tubular cells have abundant mitochondria and rely primarily on oxidative phosphorylation to obtain energy; thus, they are very sensitive to ROS-induced damage, such as mutation, apoptosis and mitochondrial-DNA-associated functional defects29. Under Rrm2b-deficient conditions, a decrease in the Aldh7a1 expression level reduced carboxylic acid and betaine production, which indirectly intercepted pathways leading to oxidative stress and osmotic stress. In response to severe stress, compensatory mechanisms could be activated in the Rrm2b deleted kidney. One such mechanism is the proline generation and accumulation through the upregulation of P5cs and Pycr. Proline has also been reported to protect against oxidative stress, and its accumulation in cells is a response to physiological stress36.Another cellular compensation mechanism is enhanced Bhmt and reduced Dmgdh expression, both of which cause dimethylglycine accumulation and homocysteine consumption in the kidney, resulting in reduction of oxidative damage due to high levels of homocysteine. Previous reports have shown that homocysteine is a toxic compound to tissues in high concentrations38. Therefore, loss of Rrm2b in cells results in elevated oxidative stress and dysregulated metabolic activities, with subsequent activation of certain alternative compensatory pathways to counteract oxidative damage.Unbalanced metabolism usually induces an overload of metabolites, accompanied by accumulation of cellular damage and overall cellular stress. Aldh7a1 protects cells against osmotic stress and thus attenuates osmotic stress-induced oxidative stress by metabolizing toxic aldehydes to produce NAD(P)H, which helps restore the cellular redox balance+-K+ ATPase pump. This study indicated that Rrm2b not only mediates mitochondrial DNA replication and repair but is also critical for the maintenance of mitochondrial functional integrity and metabolic activities. Using an Rrm2b-deficient (Rrm2b kiKO) mouse model, we propose that Rrm2b is essential for normal functions of the kidney.Mitochondria supply ATP, which is required for critical cellular functions throughout the body, especially in highly energy-dependent organs. Kidneys require a large amount of energy to filter minerals and other materials, perform tubular reabsorption and pass urine out of the body. Renal proximal tubules have abundant mitochondria to support ion transfer, such as that performed by the NaSupplemental figures and Supplemental TablesSupplemental Tables


Writing and editorial assistance was provided by Johna-than Maher, PhD, of BlueSpark Healthcare Communications, and was contracted by Boehringer-Ingelheim Pharmaceuticals, Inc. Dr. Govindan meets the criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE), was fully responsible for all content and editorial decisions, and was involved in all stages of manuscript development. Dr. Govindan received no compensation related to the development of this article.Ramaswamy Govindan has served as a paid consultant or been on the Advisory Board of AstraZeneca; Avantis Medical Systems, Inc.; Bristol-Myers Squibb Company, Boehringer Ingelheim GmbH; and Eli Lilly and Company, and is a member of the Speaker’s Bureau of Eli Lilly and Company and Genentech, Inc.


Due to increased longevity and generational location, grandparent death creates new contexts for identity, family culture, and intergenerational relationships. To explore this loss from two perspectives, we conducted intensive interviews with young adults and their mothers (N = 16) who experienced a recent grandparent (parent) death. Guided by the life course perspective, we were interested to learn how grandparent death may shape identity, meaning, and behaviors among family members, and influence their shared parent-child relationship. Findings suggest that the relationship with the grandparent, the diverse expressions of grief, the navigation of family transitions during and after death, and the curation of grandparent memories influenced individual and family outcomes. Implications suggest the need for varied supports that are sensitive to how individual family members approach grief in distinct ways reflective of their developmental positions, past experiences, and relational expectations.


Takayasu arteritis (TA) is a chronic and nonspecific inflammatory disease mainly affecting the aorta and its major branches, resulting in the stenosis or occlusion of target arteries. Approximately 50% to 60% of patients with TA likely have renal artery stenosis (RAS), which results in refractory hypertension (HTN) and renal dysfunction. Aortorenal bypass with saphenous vein graft (SVG) is the classical procedure to relieve patients’ symptoms. Graft restenosis is the most common complication during long-term follow-up. However, aortorenal vein graft aneurysm (AVGA) is uncommonly reported, and symptomatic or ruptured AVGA that needs reoperation is even rarer. Long-term follow-up results after AVGA reoperation also remain scare. Here, we introduced the long-term result of a symptomatic AVGA under the reoperation of polytetrafluoroethylene (PTFE) graft replacement and provided a literature review of AVGA reoperation after surgical bypass for RAS.An 18-year-old male complained about mild to severe right lumbar pain for 5 days. He underwent right aortorenal bypass with SVG for TA-induced right renal artery stenosis to relieve refractory HTN and renal dysfunction 2 years ago. However, this patient did not proceed with a follow-up after the procedure. Physical examination showed normal vital signs, and an obvious percussion tenderness over the right kidney region was detected. The updated computed tomography angiography (CTA) revealed a right AVGA with a maximum diameter of 26 mm. No restenosis of the proximal and distal anastomoses was detected.The patient was diagnosed to have right aortorenal vein graft aneurysm at the risk of rupture and Takayasu arteritis.The AVGA was resected with a 6 mm PTFE graft replacement. An end-to-side proximal anastomosis to the orifice of the original anastomosis on the abdominal aorta and an end-to-end distal anastomosis to the distal normal renal artery were made.The patient had an uneventful postoperative clinical course and was discharged from the hospital 5 days after the operation. The 4-year updated CTA revealed no restenosis or aneurysmal degeneration of the prosthetic graft.Symptomatic AVGA that needs reoperation is rare. Prosthetic graft replacement is an effective way to eliminate the risk of potential rupture. A 4-year satisfactory result indicative of a prosthetic graft can be the first choice for aortorenal bypass in RAS without active biological inflammation. In 1908, Takayasu, a Japanese ophthalmologist, first described this disease in a young female patient with retinal neovascularization and absent radial pulses.–8 Endovascular therapy is the preferred initial treatment because it is minimally invasive. However, previous studies about endovascular therapy showed inconsistent results regarding long-term patency.–6 In other studies, surgical bypass as an alternative has demonstrated superior patency rates to endovascular intervention. Aortorenal bypass with a saphenous vein graft (SVG) was previously the optimal option.,8 One of the most common complications of surgery is the restenosis and dilation of SVG. However, reports on aortorenal vein graft aneurysm (AVGA) after surgical repair for RAS induced by TA are few, and ruptured AVGA is even rarer, with only 2 published cases without long-term results after reintervention.,10 In this case, we first reported the long-term result of reintervention for AVGA at the risk of rupture and provided a literature review about the treatment of AVGA after surgical bypass for RAS.Indications for revascularization procedures remain unclear because limited research has reported about the prognosis of patients who have TA with renal artery stenosis (RAS) and do not undergo vascular intervention. Revascularization procedures include endovascular therapy with or without stent and surgical repair with an autologous graft or a prosthetic graft.2An 18-year-old male complained about refractory HTN for 2 years. He was under therapy involving 2 different antihypertensive medicines, and his blood pressure varied from 160/110 mm Hg to 150/100 mm Hg. Physical examination showed the following findings: temperature of 36.7°C, heart rate of 73 bpm, respiratory rate of 16 breaths per minute, and blood pressure of 162/90 mm Hg. Abdominal computed tomography angiography (CTA) revealed a severe stenosis lesion (>70%) in the middle segment of the right renal artery with obvious intimal hyperplasia in the stenosis part and slenderness of the left renal artery , and the blocked time for the right renal artery was limited to 20 minutes. The patient had an uneventful postoperative clinical course and was discharged from the hospital 4 days after the operation. One month after the procedure, eGFR increased to 95 ml/minute, and Crea remained normal. The blood pressure was well controlled by only 1 antihypertensive medicine. CTA showed that the right SVG was patent without any restenosis or dilation  graft was interposed. An end-to-side proximal anastomosis to the orifice of the original anastomosis on the abdominal aorta and an end-to-end distal anastomosis to the distal normal renal artery were made Fig. A and B. 3,8,11TA is a chronic and nonspecific inflammatory disease mainly affecting the aorta and its major branches, resulting in the stenosis or occlusion of target arteries. Approximately 50% to 60% of patients with TA likely have RAS, which results in refractory HTN, cardiac inefficiency, and renal dysfunction. The revascularization of the renal artery may be necessary when symptoms worsen. Though revascularization procedures include endovascular therapy with or without stent deployment and surgical repair, studies have reported that surgical revascularization has a long-term patency and a lower risk of restenosis compared with those of endovascular therapy. Otherwise, surgical repair is more suitable for long segments and severe stenosis and have a broader indication than endovascular therapy.–17 In 1973, Stanley first reported the follow-up results of 100 cases of aortorenal SVG, indicating that SVG has a higher likelihood of dilation or aneurysmal degeneration in a pediatric population; a long-term follow-up is necessary for the assessment development of SVG. However, in a review about the complications of renal artery reconstruction that requires reoperation, the aneurysmal deterioration of vein grafts is an uncommon reason, accounting for only 3 (1%) of 278 aortorenal saphenous vein bypass grafts requiring revision. Dean also reported that about 20% (8/39) of the aortorenal SVG dilates, whereas AVGA is detected in only 2 patients without growth during follow-up. In a report on anastomotic aneurysms after the surgical repair of TA, the incidence of anastomotic aneurysm in 103 patients with surgically treated TA is 8.5%. However, aneurysm degeneration involves anastomosis, not saphenous vein bypass. Otherwise, the involved arteries are the aorta, the subclavian artery, and the carotid artery; furthermore, 25% lesions of aneurysms in TA indicate that aneurysmal TA is an independent predictor of the development of an anastomotic aneurysm.The reconstruction of the renal artery with the saphenous vein and a prosthetic material as a bypass graft is durable for atherosclerotic disease. However, the risk of SVG dilation in pediatric patients and patients without atherosclerosis remains unresolved. For the long-term patency of an aortorenal bypass graft, most studies have focused on SVG complications or prosthetic graft restenosis and SVG dilation. Limited studies have described AVGA, especially symptomatic or ruptured AVGA. Rarer cases of AVGA reoperation and long-term results after re-intervention have been reported because most cases of AVGA remain stable during follow-up and are uneventful.,19 Other cases of AVGA after surgical bypass for RAS are either under observation without reoperation or under reoperation without follow-up after discharge. Our case underwent a 4-year follow-up with a satisfactory result. We searched relevant information about reoperations for AVGA after the surgical bypass of RAS via PubMed. The detailed information of the related studies is shown in Table The aneurysm formation of SVG in non-aneurysmal renal artery lesion caused by TA is extremely rare. Cases requiring a reoperation are even rarer, and only 2 cases provided 1- and 2-year follow-up results after the second intervention.–20 We also adopted PTFE graft replacement. Excluding the PTFE graft, Bath reported the replacement of a previous AVGA by using a Dacron graft in 2012. In contrast to our procedure. An alternative autologous graft to SVG has also been proposed. Patterson reported the reconstruction of the renal artery with the radial artery and documented a satisfactory result in a case. Excluding surgical repair for AVGA, Hu also introduced a novel endovascular intervention for AVGA with a cover stent and coils. Berkowitz adopted a new way to prevent the risk of the aneurysmal degeneration of SVG. Our results showed that a tubular SVG supported by an external Dacron mesh could be a suitable graft material for renal reconstruction in a pediatric population. With an average follow-up of 4.3 years, no AVGA was detected in 19 mesh-supported grafts.The most common way to solve AVGA is AVGA replacement by using a prosthetic graft. Five cases of AVGA were successfully resolved by aneurysm resection with an interposed prosthetic graft . However, only 1 case documented a 1-year follow-up with a satisfactory result; 4 other cases did not reveal good results after discharge.Considering the satisfactory result after the 4-year follow-up, we believe that prosthetic graft may be the first choice of graft for patients who have TA-induced RAS and need to undergo aortorenal bypass if no active biological inflammation is detected because biological inflammation increases the likelihood of complications after aortorenal bypass in patients with TA.AVGA after surgical bypass with SVG for TA-induced RAS is uncommon. Symptomatic AVGA requiring a reoperation is even rarer. Prosthetic graft replacement is an effective way to eliminate the risk of potential rupture. However, the long-term result of reoperation for AVGA with prosthetic graft is still scarce. Our case demonstrated a satisfactory 4-year follow-up prosthetic result without restenosis or anastomotic aneurysmal formation, indicating that prosthetic graft might be considered as the first choice for RAS if no active biological inflammation was detected.Conceptualization: Jichun Zhao.Data curation: Bin Huang.Resources: Yi Yang.Software: Yi Yang.Visualization: Ding Yuan.Writing - original draft: Xiyang Chen.Writing - review & editing: Bin Huang, Jichun Zhao.


ISA2) was up-regulated by HT. Actually ISA2 was the only gene up-regulated by all three stress treatments. The expression of 17 genes was up-regulated, while six genes, including granule-bound starch synthase (GBSSI), AGPS2, BEIII, PHOL, ISA1, and AGPL2, were down-regulated by DS. Eleven genes were down-regulated and 12 were up-regulated by HT+DS. The activity of ADP-Glc pyrophosphorylase, starch synthases, GBSS, SS, and starch branching enzymes in the stress treatments  often appeared to peak values in advance and declined significantly to be lower than that in the CK. The genes that coordinated participation in the enzymes formation can serve as an indicator of the enzymes activity potentially involved in starch biosynthesis. HT, DS, and HT+DS altered the timing of starch biosynthesis and also influenced the accumulation of amylose, amylopectin, total starch, and sucrose. Under HT, DS, and HT+DS, the key enzymes activity and their genes expression associated with the conversion of sucrose to starch, was reduced, which was the leading cause of the reductions in starch content. Our study provide further evidence about the effects of stress on starch biosynthesis in wheat, as well as a physiological understanding of the impact of post-anthesis heat and DS on starch accumulation and wheat grain yield.High temperature (HT) and drought stress (DS) play negative roles in wheat growth, and are two most important factors that limit grain yield. Starch, the main component of the wheat [endosperm, accounts for 65–75% of grain weight, and is significantly influenced by environmental factors. To understand the effects of post-anthesis HT and DS on starch biosynthesis, we performed a pot experiment using wheat cultivar “Zhengmai 366” under field conditions combined with a climate-controlled greenhouse to simulate HT. There were two temperature regimes (optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C from 10 days after anthesis to maturity) accompanied by two water treatments . Optimum temperature with optimum water treatment was the control (CK). We evaluated the expression patterns of 23 genes encoding six classes of enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. HT, DS, and HT+DS treatments altered gene expression profiles. Compared to the CK, expression of 22 of the 23 genes was down regulated by HT, and only one gene ( Triticum aestivum L.), grown in many different environments worldwide, is one of the major cereal crops and an important source of calories for human nutrition. Environmental constraints are major influence factors for wheat productivity in many regions. Wheat is particularly susceptible to temperatures above the range of 20–30°C, and mean temperatures during the wheat grain-filling stage often exceed the optimum in most wheat-growing regions, which is considered to be one of the most important factors limiting wheat grain yield (Wheat (in yield . Some whin yield . Becausein yield . Wheat iin yield . Droughtin yield .ADP-Glc pyrophosphorylase (AGPase) and granule-bound starch synthase (GBSS). Amylopectin is mainly synthesized by the coordinated actions of AGPase, starch synthases (SSs), starch branching enzymes (SBEs), and starch debranching enzymes (DBEs) (PHO), metabolizing sucrose to the carbon precursors, is also needed for starch biosynthesis  . In addiynthesis . There aand GBSS . Both hiand GBSS . Temperaand GBSS .SSIIb, SSIIc, SSIIIb, SSIV, BEIIb, BEIII, GBSSII, ISAI, ISA2, PUL, PHOH, and PHOL, have not yet been examined under conditions of high temperature and water deficit. Furthermore, most studies examine the effects of either high temperature or drought stress seperately, thus give insufficient knowledge of the combined effects of high temperature and drought stress. Therefore, in this study, we analyzed the relative transcription of 23 isozyme genes encoding enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. We also evaluated the activities of starch biosynthetic enzymes, and the accumulation of starch and sucrose. Furthermore, we simulated the starch biosynthesis process in response to high temperature and drought stress. The expected results will enable a better understanding of the influence of high temperature, drought stress, and their interaction on starch biosynthesis, and will also extend our knowledge on the impact of post-anthesis heat and drought stress on starch synthesis and grain yield in wheat.High temperatures and drought stress, as the major abiotic stress factors that affect wheat growth, development, and production , occur f2O5, and 1.15 g K2O per pot before sowing. Pots were buried in the field, keeping the soil surface inside the pot at the same level as that of the field. The trial was arranged in a randomized complete block design with three replicates. The plants were thinned to 12 seedlings per pot at the three-leaf stage. An additional 1.1 g N per pot was then added at the stem elongation stage.Seeds of “Zhengmai 366,” a widely cultivated local wheat variety, were obtained from the Henan Academy of Agricultural Sciences. An experiment combining pot culture with a climate-controlled greenhouse to simulate high temperature was conducted at the farm of the Scientific and Educational Station of Henan Agricultural University, Zhengzhou, China  during the 2015–2016 and 2016–2017 growing seasons. Seeds were sown in 24 cm-diameter pots containing 10 kg loam soil. The soil was collected from the top 20 cm soil layer at the experimental site. The total N, rapidly available phosphorus, and potassium were 1.43, 68.14, and 204.87 mg/kg, respectively. Fertilizer was applied at a rate of 1.15 g N, 1.35 g PThe spikes were tagged for sampling when the anthers had extruded from the florets in 50% of the tillers in each pot. The plants in pots were grown under field conditions until they were transferred to the climate chamber for high temperature treatment beginning at 10 days after anthesis (10 DAA). In the controlled climate chambers, two temperature regimes consisting of optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C were accompanied by two water treatments; an optimum water treatment with ∼75% of the relative soil water content, and a mild drought stress treatment with ∼50% of the relative soil water content, was set to wheat maturity. The optimum day/night temperature combined with the optimum water treatment was the control (CK), and the plants were irrigated regularly to maintain the soil relative water content at ∼75%. High day/night temperature with optimum water treatment was defined as the high temperature (HT) treatment. For the drought stress (DS) treatment, the water content was controlled from ∼3 DAA prior to transfer to the climate chambers to ensure that the soil relative water content was maintained at the objective level of ∼50%. The soil water content was determined by weighing the pots, combined with time domain reflectometry measurements . The treatment consisting of high day/night temperature and DS was defined as the combined HT and drought stress (HT+DS) treatment.Tagged heads were initially collected before plants were transferred to the climate chamber. Sampling was then performed every 4 days until seed maturity. One portion of the grains sampled were frozen immediately in liquid nitrogen for 30 min and then stored at °80°C for RNA extraction and cDNA synthesis as well as for determination of the activity of starch biosynthesis enzymes, while another portion of the grains sampled were dried to constant weight for measurement of sucrose, soluble sugar, and starch contents as well as for determination of the starch accumulation rate. All the determination was in triplicate for each biological replicate and each sampling date contained three biological replicates.RNA was extracted from wheat grains at each sampled time point during grain development. Total RNA from grain endosperm was extracted using a test kit from Takara and purified with gDNA Eraser (Takara) to remove contaminating genomic DNA. cDNA was synthesized from 2 µg of total RNA using the ThermoScript Reverse Transcriptase Reagent Kit (Takara). The integrity of the RNA samples was examined by gel electrophoresis.Expression profiles of the wheat genes were determined by quantitative real-time PCR analysis. cDNAs for all of studied genes were amplified using primers designed from known wheat nucleotide sequences (SS), GBSS, soluble starch synthase (SSS), AGPase, and starch branching enzyme (SBE), respectively, using the reagent kits SS-2-Y, GBSS-2A-Y, SSS-S-Y, AGP-2A-Y, and SBE-2-Y obtained from Su Zhou Keming Biotechnology Incorporated. The activities of GBSS, SSS, and AGPase were determined as nmol min–1 of NADPH produced g–1 grain FW; SS activity was measured by quantifying the production of sucrose from fructose and glucose (µg min–1 g–1 grain FW); and SBE activity was determined as the percentage decrease in absorbance at 660 nm of that decrease of iodine blue value as an enzyme activity unit.We assayed the activities of five starch biosynthesis enzymes, including starch synthase (The contents of soluble sugars and sucrose in wheat grains were determined using the anthrone colorimetric and resorcinol colorimetric methods as described by The amylose and amylopectin contents in wheat grains were determined with a coupled spectrophotometric assay as described by Determination of starch accumulation rate was referenced to the method of http://bioinformatics.psb.ugent.be/webtools/Venn/).Data from three biological replicates in the experiment were statistically analyzed for variance and correlation using SPSS 21.0. The line graphs were prepared and edited with SigmaPlot 13.0. Venn diagrams of gene expression were obtained using the Bioinformatics & Evolutionary Genomics online analysis software  expression in DS treatment peaked by 4 days (apart from BEIII by 8 days) in advance, while AGPS1-b and SSIIIa expression peaked by 4 days late. For the HT, HT+DS treatments, the genes expression, including SSIIb, SSIIc, SSIIIb, SSIV, BEI, BEIII, PUL, and PHOH, peaked by 4 or 8 days earlier than the CK. However, the expression of genes  in HT treatment, and the expression of genes  in HT+DS treatment, all had the maximum values at 10 DAA, and showed gradually declining trend during grain filling stage.In the CK, the expression of 23 genes all displayed a unimodal curve with the peak times varying for the different genes Figure 1.AGPS2, AGPL2, GBSSI, ISA1, PHOL while significantly increased the expression of AGPS1-a, AGPS1-b, AGPL1, BEI, BEIIb, ISA2, PUL, PHOH; HT significantly reduced the expression of AGPL1, AGPL2, AGPS1-a, AGPS1-b, AGPS2, SSI, SSIIa, SSIIc, SSIIIa, SSIIIb, ISA1, ISA2, PHOL; HT+DS significantly reduced the expression of AGPL1, AGPL2, AGPS1-a, AGPS1-b, AGPS2, SSIIa, SSIIIa, SSIIIb, ISA1, PHOL, while only led to the increase of ISA2 expression  showed up-regulated expression; ISA2 was the only gene in which expression was up-regulated by the HT, DS, and HT+DS treatments  was down-regulated  and the CK, the positive and negative values represent up-regulated and down-regulated gene expression, respectively. Of the 23 genes examined, relative expression in 22 was down-regulated by HT  with the expression of their isozyme genes as a whole at 22–26 DAA.The expression of isozyme genes is obviously related to the activities of the enzymes they encode during different phases of grain filling, especially at the late stage (The expression of genes for isozymes involved in starch biosynthesis was closely correlated with the contents of starch and its components (amylose and amylopectin) and with the contents of sucrose and soluble sugars. The expression of genes involved in starch biosynthesis mainly affected the accumulation of amylopectin and total starch, and the expressions of most of the 23 genes showed significant correlations (p < 0.05 or p < 0.01) with amylopectin and total starch content in most of the different sampling periods Table 4.SS activity with the amylopectin and starch content were significant (p < 0.05 or p < 0.01) in different sampling times. The correlations of SBE and AGPase activities with the amylopectin and starch content were significant (p < 0.01) from 22 to 26 DAA. The correlations of GBSS and SS activities with the amylopectin and starch content were significant (p < 0.05 or p < 0.01) except for 18 DAA. The SSS activity was more closely related to the dynamic accumulation of amylose, amylopectin, and total starch than the other key enzymes. And the activities of starch biosynthetic key enzymes were more closely related to the amylopectin and total starch content than to the amylose content, sucrose content, and soluble sugar content. The correlations between the activities of starch biosynthetic key enzymes and starch accumulation rate were significant (p < 0.05 or p < 0.01) (except for SBE and AGPase activity at 14 DAA). Which indicated that the activity of starch biosynthetic key enzymes directly influenced starch accumulation in wheat endosperm.The enzyme activities during different phases of grain filling were found to be closely related to the dynamic accumulation of starch and sucrose Table 5.AGPase, SSS, GBSS, and SBE, all of which are key enzymes associated with the conversion of sucrose to starch, were reduced, and this could explain the decline in starch accumulation. Transcription of the genes encoding AGPase isoforms  was reduced by the HT and HT+DS treatments, leading to the repression of the conversion of sucrose from glucose-1-phosphate (G-1-P) to ADP-glucose (ADPG). The expression of all the main genes involved in the conversion of ADPG to amylopectin  was reduced to some extent in response to HT, DS, and HT+DS, although the expression of ISA2 increased. This led to a significant decrease in the amylopectin content and was also the primary cause of the decrease in total starch content. Under DS, the activities of AGPase, SSS, and SBE were reduced, although the expression of AGPS1-a, AGPS1-b, AGPL1, BEI, BEIIa, BEIIb, and SS isozyme genes increased, which implied that starch biosynthetic enzymes did potentially display the coordinated participation of many genes not depend on one or some genes expression. The conversion of sucrose from G-1-P to ADPG and of ADPG to amylopectin was also probably repressed, leading to a significant reduction in the amylopectin and starch content. In the HT, DS, and HT+DS treatments, the decrease in starch accumulation was strongly associated with the change in starch biosynthesis in the wheat grain endosperm, which is attributed to the dynamic change in the activities of key enzymes and the expression profiles of their isozyme genes involved in starch biosynthesis.The simplified model of the starch biosynthetic pathway was demonstrated (PUL and up-regulation of SSIIIb expression in response to HT was previously observed in barley (ISA2) expression was down-regulated by HT, while of the 23 genes expression, 17 was up-regulated by DS. For the HT+DS treatment, almost half-and-half of the 23 genes expression was down-regulated and up-regulated, respectively. High temperature significantly decreased the relative expression of the 23 genes and the negative effect on genes expression was greater than DS.There are few reports which describe the expression of genes involved in starch synthesis in wheat under high temperature and DS , althougn barley . High ten barley . In the SS was found to be the most sensitive enzyme in starch biosynthesis to drought. The activity of SS is highly correlated with starch synthesis rate -linked glucan primer is catalyzed by SSs  in wheat  between HT and DS.The results of our study showed that the HT, DS, and HT+DS treatments caused earlier maturation of wheat by 8, 4, and 12 days, respectively, compared with the CK. During the grain filling stage, the dynamic change in the expression of genes and enzyme activity involved in starch biosynthesis in wheat endosperm was all altered, and often appeared to peak values earlier. Of the 23 genes expression, 22 was negatively influenced by HT, while only 6 and 11 was down-regulated by DS and HT+DS, respectively. The expression levels of genes encoding enzymes involved in starch biosynthesis can possibly serve as indicators of the activities of starch biosynthetic enzymes during grain filling. The activity of HL and CW conceived the original research plan. YH and WL performed most of the experiments and analyzed the data. DM and QH supervised the experiments. YH and GM performed the real-time qPCR analysis. HL wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Context: Oxymatrine (OMT) has various pharmacological effects, including immune reaction regulation, anti-inflammation and anti-hypersensitive reaction.Objective: This is the first report to investigate the molecular mechanism of OMT function in l-arginine (Arg)-induced acute pancreatitis (AP) involving intestinal injury.Materials and methods: Rat pancreatic AR42J and small intestinal IEC-6 cells were treated with Arg (200–800 µM) for 48 h plus OMT (4 mg/mL) treatment. Thirty adult Wistar rats were randomly assigned to control , AP (i.p. of 250 mg/100 g body weight Arg) and OMT (i.p. injection of 50 mg/kg b.w. OMT every 6 h following Arg). Both cells and rats were harvested at 48 h.Results: Arg-induced cell proliferation in both rats AR42J (EC50 633.9 ± 31.4 µM) and IEC-6 cells (EC50 571.3 ± 40.4 µM) in a dose-dependent manner, which was significantly inhibited by OMT (4 mg/mL). Meanwhile, Arg (600 µM) induced expression of proinflammatory cytokines  and activation of p-p38/p-ERK in vitro, which was reversed by OMT. In vivo, OMT (50 mg/kg) inhibited 250 mg/100 g of Arg-induced AP involving intestinal injury, including inhibiting Arg-induced inflammatory in pancreas and intestine, inhibiting Arg-induced increase of TNF-α, IL-6, IL-1β, NF-κB and p-p38/p-ERK-MAPK signalling, and inhibiting Arg-induced increase of IL-17A/IL-17F, IFN-γ, ROR-γt and T-bet. Meanwhile, OMT inhibited Arg-induced expression of CD44 and CD55 in intestinal injury.Discussion and conclusions: OMT protects against Arg-induced AP involving intestinal injury via regulating Th1/Th17 cytokines and MAPK/NF-κB signalling, which is a promising therapeutic agent in clinics. Acute pancreatitis (AP) is not only a local pancreatic inflammation but also a systemic disease involving multiple organs. The intestinal barrier dysfunction plays a pivotal role in AP progression  through mediating inflammation and Treg/Th17 in CIA rats , a semi-essential amino acid in protein synthesis, plays a significant role in regulating immune response, hormone secretion and wound healing . The animals were kept from the specific pathogen-free (SPF) Animal Experimental Ministry of China Medical University. All animals were housed in plastic cages containing wood shaving and maintained in a room at 25 °C with a 12 h light/night cycle with free access to standard laboratory diet and water. Animals were maintained according to institutional regulations in facilities approved by the Animal Care Committee of China Medical University in accordance with Chinese government guidelines for animal experiments.n = 5): control  and OMT (OMT treatment). The dose of Arg and OMT follows our previous study : control , AP (Arg treatment) and OMT (OMT treatment after Arg induction). As described previously  were cultured as previously described  for 48 h with or without OMT (4 mg/mL) treatment for the last 24 h. The optimum concentration of Arg (600 µM) and OMT (4 mg/mL) was calculated from our pre-experiment (MTT assays shown in the ‘Results’ section). Cells were starved at 2 h with serum-free media prior to incubation with Arg or OMT.50 to calculate the drug function in Arg and Arg plus OMT-treated groups, respectively.AR42J and IEC-6 cells growth under the various doses of Arg combing with or without OMT (4 mg/mL) was detected by MTT. Briefly, cells were harvested, counted and then seeded into 96-well plates at the density of 8000 viable cells per well overnight. After starvation (2 h), Arg was added to the media with various concentrations shown in the ‘Results’ section. Cells were incubated with 10 µL of MTT  for 4 h at 37 °C. The media was then removed and 150 µL of dimethyl sulphoxide (Sigma) was added to each well. Each experimental plate was read on ELISA 96-well microtiter plate reader  at 570nm wavelength. Experiments were performed in triplicates, and data were presented as the percentage of treated cells compared with control cells. We using half maximal effective concentration ECTerminal ileum specimens (5 cm) were stained with haematoxylin and eosin (HE) and blindly examined under microscopy by three special pathologists. As described previously . Each experiment was repeated three times consecutively.Total RNA was extracted from AR42J and IEC-6 cells and intestinal tissue samples from control, AP and OMT groups with TRIZOL reagent under the manufacturer . The expression of target genes was analyzed in a light cycler 2.0 with the light cycler kit (Takara). The conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers are summarized in Total protein lysates were prepared from AR42J and IEC-6 cells and intestinal tissues from control, AP and OMT groups. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes  and incubated with primary NF-κB , IκBα , p-p38/p38 , p-ERK/ERK , ROR-γt , T-bet  and GAPDH  antibodies overnight at 4 °C. Immunoreactive protein bands were visualized with an ECL detection kit . Each experiment was repeated three times.2O2 for 20 min. Nonspecific antibody binding was then blocked using a specific blocking reagent for 30 min. CD44 (Abcam) and CD54 (Abcam) antibodies were incubated overnight at 4 °C. The corresponding secondary antibodies were incubated at room temperature for 20 min. Reaction products were visualized by incubation with 3,3′3-diaminobenzidine and then counterstained with haematoxylin. Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). Staining extent was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%) according to the percentage of the injury involved area as previously described  was performed as described previously . The differences of MTT, qRT-PCR and western blot (WB) assays were expressed as mean ± SE and compared through Student’s In normal rat pancreatic AR42J cells and intestinal IEC-6 cells, OMT (4 mg/mL) treatment had no effect in pro-inflammatory cytokines  mRNA expression compared with control group. IL-17A, IL-17F and IFN-γ, as the symbolic cytokines secreted by Th1 and Th17 cells, were unchanged as well . MeanwhiIn vivo, there was no difference in the morphology of both pancreas and intestine between OMT and control groups  significantly inhibited Arg-induced cell growth, especially at 600 µM of Arg treatment (p < 0.01) (in vitro.MTT showed that Arg treatment (48 h) enhanced cell proliferation in a dose-dependent manner in both AR42J and IEC-6 cells . The EC5 < 0.01) . Based oIn pancreatic AR42J cells, Arg (600 µM) significantly induced pro-inflammatory cytokines TNF-α, IL-6 and IL-1β mRNA expression. Meanwhile, IL-17A, IL-17F and IFN-γ were also upregulated by Arg (600 µM). However, OMT (4 mg/mL) significantly reversed the increase of above cytokines induced by Arg , compareWB showed that Arg (600 µM) significantly induced NF-κB, p-p38 and p-ERK protein expression. However, OMT (4 mg/mL) reversed Arg-induced increase of NF-κB, p-p38 and p-ERK protein levels , comparein vitro.Taken together, OMT inhibits Arg-induced inflammation and MAPK/NF-κB signalling Arg (250 mg/mL) successfully induced different severities of AP in 10 rats combined with the disruption of the intestinal barrier in histopathology and morphology assays .The control group exhibited normal histological features of the pancreas  .Histology of intestinal tissue from the control group rats revealed normal mucosal architecture with similar histologic scores . The AP Taken together, OMT attenuates Arg-induced pancreatic and intestinal histologic damage.Arg (250 mg/mL) group showed a significant increase of TNF-α, IL-6, IL-1β, IL-17A, IL-17F and IFN-γ mRNA levels in intestinal tissues, compared with control group. However, OMT (50 mg/kg) reversed the increase of above inflammatory cytokines induced by Arg, compared with AP group .The Arg (250 mg/mL) group showed a significantly increase of NF-κB, p-p38 and pERK protein levels in intestinal tissues compared with the control group. However, the increase of the above proteins induced by Arg in AP group was partially reversed by OMT (50 mg/kg) .Since ROR-γt and T-bet are signature transcription factors and regulators in the differentiation and cytokines release of Th1 and Th17 cells, we further investigated OMT function in regulating ROR-γt and T-bet activity. WB showed that Arg significantly induced ROR-γt and T-bet protein expression, which were also reversed by OMT treatment .p < 0.01; p < 0.01, respectively). However, OMT (50 mg/kg) partially inhibited Arg-induced CD44 and CD54 protein expression in the AP group .IHC showed that both CD44 and CD54 were localized in the cytoplasm and membrane in intestinal tissues . A signiin vitro and in vivo.OMT exhibits a protective effect in multiple agent-induced injuries in different animal models, including liver, heart, brain and intestine  and p38/ERK-MAPK signalling. OMT also protects other agent-induced intestinal injury. OMT protected against cirrhosis-associated intestinal mucosal damage via inhibiting NF-κB-mediated signalling and attenuated intestinal ischaemia/reperfusion injury in rats via inhibiting TNF-α and p-p38/MAPK signalling  expression in vitro and in vivo via regulating pro-inflammation cytokines and mediators, MAPK signalling, Th1/Th17 cytokines and corresponding transcripts factors ROR-γt and T-bet expression. OMT is considered as a potential therapeutic agent in AP and intestine injury.As shown in


Acinetobacter baumannii strain was isolated from hospital wastewater and used as the donor in a filter mating experiment with an Escherichia coli strain as the recipient. Recipient, donor, and transconjugant were sequenced, and both donor and transconjugant were found to harbor highly similar plasmid sequences, suggesting that plasmid transfer had occurred.A ceftazidime-resistant  Acinetobacter baumannii strain was isolated from hospital wastewater and used as the donor in a filter mating experiment with an Escherichia coli strain as the recipient. Recipient, donor, and transconjugant were sequenced, and both donor and transconjugant were found to harbor highly similar plasmid sequences, suggesting that plasmid transfer had occurred.A ceftazidime-resistant  Acinetobacter baumannii strain, WPB102, which was isolated from hospital wastewater effluent (Escherichia coli SCC1 strain (WPB103) was used as the recipient (600] = 0.1) were washed twice and mixed in equal volumes. An aliquot of 100 µl of the mixture was loaded onto a 0.45-µm cellulose nitrate membrane (Sartorius Stedim) and placed onto LB agar for incubation at 37°C for 16 h. Biomass on the membrane was dislodged, serially diluted, and plated on agar supplemented with antibiotics to select for recipient, donor, and transconjugant, namely, chloramphenicol (15 ppm) in tryptone bile X-glucuronide (TBX) agar, ceftazidime (8 ppm) in LB agar, and chloramphenicol (15 ppm) plus ceftazidime (8 ppm) in TBX agar, respectively. The plates were incubated at 37°C for LB agar and 44°C for TBX agar. The transfer efficiency, calculated as the ratio of transconjugants to recipients, was 1.81 × 10−3 ± 3.25 × 10−4. Antibiogram results from the AST-GN79 panel confirmed that the transconjugant (WPB121) isolated from the selective medium was resistant to ceftazidime.Antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) can act as agents for the selection and transfer of antimicrobial resistance –6. To de–effluent , was seleffluent . Phenotyecipient . Conjugaecipient . OvernigN50 values of 188,313, 150,429, and 178,426 bp. Plasmid and chromosome sequences were predicted using PlasFlow 1.1 with default settings . Genomic DNA was prepared using an NEBNext Ultra DNA library prep kit  and sequenced on the Illumina HiSeq 4000 platform to obtain 150-bp paired-end reads. Totals of 7.1, 6.8, and 7.1 million paired-end reads were assembled using VelvetOptimiser 2.2.4  with a msettings . DefaultPlasmid sequences in donor and transconjugant genomes were compared using NCBI BLASTn and were found to share 99% nucleotide similarity and 99% alignment coverage, providing evidence of plasmid-transferred ceftazidime-resistant genes and interspecies transmission of clinically relevant ARGs.CP034426 to CP034428. Raw reads can be found in the NCBI Sequence Read Archive (SRA) under the accession numbers SRR8289913 to SRR8289915.Whole-genome sequences of the transconjugant, recipient, and donor were deposited in GenBank under the accession numbers


DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA) circuit to initiate a hybridization chain reaction (HCR) circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal. Enzyme-free DNA circuits  that reli.e., ELISA assays) have proven extremely successful; for example, the 96-well Neutr-avidin plate used herein has already been widely proofed in assay development [CHA has proven to be particularly good at staged amplification, while HCR is particularly good at amplification on surfaces such as electrodes or magnetic beads ,9,10,11.elopment ,13.In our conception , a singlThe coupled reaction in fact showed coupled amplification, indicating the relative simplicity of ganging together nucleic acid circuits for diagnostic applications. In order to further demonstrate the versatility of the reaction, we transduced the initial signal into a fluorescent reporter, a G-quantet-hemin DNAzyme.Catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR) are nucleic acid circuits in which kinetically trapped conformers can react in the presence of a single-stranded sequence-specific trigger or catalyst to form new, more energetically stable conformers. While each reaction starts from two hairpin substrates, CHA yields many duplexes, while HCR yields a double-stranded concatamer.In order to combine these two circuits, such that CHA would trigger HCR, we first had to position the single-stranded catalyst for HCR on the CHA product A. The CHThe sequences of the CHA substrates were modified so that the free 3’ end tails on H1 (2*-5*) and H2 (4*-6) could participate in other hybridization and/or strand-exchange reactions. The 16-residue tail on H1 allows hybridization of the double-stranded CHA product to an antisense sequence to the 16-residue tail (Biotin-antisense) modified streptavidin-coated plates. Since domain 2* is hidden in the initial H1 hairpin, the substrates should not efficiently hybridize to the plates. Single-stranded domain 4*-6 will initiate the HCR. The HCR circuit was designed based on the principles set out in . The two18 tails that could hybridize to a polyA18-G-quartet-hemin complex. The G-quartet-hemin complex is a well-known horseradish peroxidase-like DNAzyme that can catalyze the oxidation of p-hydroxyphenylacetic acid (HPA) to the fluorescent product bi-p,p-4-hydroxyphenylacetic acids (Bi-HPA) in the presence of H2O2. The measured emission at 410 nm should reflect the presence and concentration of the catalyst strand C1.The ultimate formation of the HCR product was monitored by including fluorescein at the 3' ends of H3 and H4. However, the coupled CHA-HCR cascade should be comparable with a wide range of reporters ,15,16,17The performance of CHA and HCR circuits was separately verified by native polyacrylamide gel electrophoresis (PAGE), which could reveal what products were being formed as the reaction progressed. For the CHA reaction, after 3 h of incubation with 10 nM C1, 200 nM H1, and 400 nM H2, a single high molecular weight product corresponding to H1:H2 appears, while the band corresponding to H1 almost completely disappears [The performance of the CHA circuit could also be confirmed by including an additional substrate, which upon interaction with H1:H2 would lead to strand displacement and release of a FAM-labeled oligonucleotide from a quencher-labeled antisense molecule. As shown in Figure S1, both the C1-dependent catalytic reaction and the background leakage were consistent with the PAGE results A. Since n) are produced in the presence of the InM. Unsurprisingly, the greater the amount of InM the shorter the concatamers produced, since the same amount of substrate is being simultaneously added to multiple, growing HCR chains. The HCR substrates were almost consumed within 5 h , and the fluorescence of the coupled CHA-HCR cascade could therefore generally be readily monitored after an overnight reaction.To independently proof the HCR reaction, an initiator mimic  was used to initiate the reaction. For assaying analytes, the coupled system was transferred onto microtiter plates. As in To confirm that both CHA and HCR were contributing to analytical sensitivity, two control experiments are carried out . First, Even greater amplification was limited by the unexpectedly high blank signal (no C1). This signal likely results from CHA leakage, HCR leakage, and non-specific binding to the plate. 2O2, and have been widely used as protein-free signal amplifiers and reporters in biosensors [As well known, some G-quartets can bind hemin and thereby form a peroxidase DNAzyme ,19 G-quaosensors ,19. We t18 tails that could hybridize to a polyA18-G1-hemin DANzyme  unless otherwise indicated. All oligonucleotides were ordered from Integrated DNA Technologies . Oligonucleotide sequences are summarized in Each well was washed three times with 200 μL of wash buffer. Some 100 μL of 1 μM Biotin-antisense in 1×TNaK  was added, and the immobilization reaction was incubated at 37 °C for 2 h. The hairpins H1 and H2 were heated to 90 °C for 5 min followed by slowly decreasing the temperature to 25 °C at a rate of 0.1 °C/s in order to ensure proper folding. Folded hairpins were incubated with different concentrations of C1 at 37 °C for 1.5 h. Each well was washed three times with 200 μL wash buffer before adding the CHA reaction. The CHA products then bound to the Biotin-antisense following incubation at 37 °C for another 1.5 h. Unreacted oligonucleotides were removed by gently washing the plates three times with 200 μL wash buffer, and the HCR reaction was initiated by adding 100 μL of 100 μM H3 and H4 (the HCR system). The plate was kept at 25 °C overnight before measurement or further reaction. 2O2; 30 nM Hemin; 0.05% Triton; 200 mM Tris, pH 8.5; 200 mM NaCl; 200 mM KCl) was added. The plate was then transferred to a TECAN Safire plate reader for fluorescence measurements with an excitation wavelength of 310 nm and emission wavelength of 410 nm.A G-quadruplex that could hybridize to the single-stranded tails of H3 and H4 was added to wells where HCR was carried out. After adding 100 μL 100 μM of a previously utilized G-quartet, EAD2 (G1)  and incuIn conclusion, we successfully built a multi-amplifier that utilized three different nucleic acid components: hairpins that executed CHA, hairpins that executed HCR, and G-quadruplex peroxidases. The primary achievement is that nucleic acid circuits can be readily mixed and matched due to the extraordinary programmability provided by base-pairing interactions. However, these results also demonstrated some of the current limitations of nucleic acid conformational transduction as an amplifier mechanism. Because of either false triggering due to the presence of lesions in the hairpins or because of thermal equilibration of the hairpin structures, the background for each layer was relatively high, and the overall amplification seen was relatively low (100-fold comparable to no amplification). While a low limit of detection could be achieved, quantitation proved difficult. The chief advantage of this analytical method is its simplicity and compatibility with surface immobilization. Unlike polymerase-based reactions, which must generally be constructed from multiple components in order to avoid the degradation of reagents or the production of false amplicons, the nucleic acid hairpins are immediately ready for use and must merely be added to an existing sample or surface. Coupled CHA-HCR circuitry can therefore potentially be adapted for the sensitive ‘yes/no’ detection of nucleic acid analytes captured from lysates in point-of-care settings. Such circuitry may also prove to be an interesting intermediate for the ligand-dependent immobilization of nucleic acid-enzyme conjugates, providing basal amplification for ELISA-like assays.


Gaps persist in HIV testing for children who were not tested in prevention of mother-to-child HIV transmission programs. Oral mucosal transudate (OMT) rapid HIV tests have been shown to be highly sensitive in adults, but their performance has not been established in children.Antiretroviral therapy-naive children aged 18 months to 18 years in Kenya and Zimbabwe were tested for HIV using rapid OraQuick ADVANCE Rapid HIV-1/2 Antibody test on oral fluids (OMT) and blood-based rapid diagnostic testing (BBT). BBT followed Kenyan and Zimbabwean national algorithms. Sensitivity and specificity were calculated using the national algorithms as the reference standard.A total of 1776 children were enrolled; median age was 7.3 years (interquartile range: 4.7–11.6). Among 71 children positive by BBT, all 71 were positive by OMT : 94.9% to 100%]). Among the 1705 children negative by BBT, 1703 were negative by OMT (specificity: 99.9% [95% CI: 99.6% to 100.0%]). Due to discrepant BBT and OMT results, 2 children who initially tested BBT-negative and OMT-positive were subsequently confirmed positive within 1 week by further tests. Excluding these 2 children, the sensitivity and specificity of OMT compared with those of BBT were each 100% .Compared to national algorithms, OMT did not miss any HIV-positive children. These data suggest that OMTs are valid in this age range. Future research should explore the acceptability and uptake of OMT by caregivers and health workers to increase pediatric HIV testing coverage. In addition, oral mucosal transudate (OMT) rapid HIV tests allow for sample collection that is less invasive, are more acceptable to clients, poses fewer risks to health care workers (HCW), and may increase testing uptake.10World Health Organization (WHO) recommendations endorse rapid antibody-based HIV tests for diagnosis of individuals >18 months.11 In 2016, the OraQuick HIV Self-Test received WHO prequalification and it is now recommended by WHO as a screening test for HIV.12 OMT has high sensitivity and specificity in detecting HIV antibodies in adults and older adolescents.10 A meta-analysis comparing OMT with BBT in adults reported a pooled sensitivity of 98.0% and specificity of 99.7% for OMT.10 OMT has not been validated in children.The Food and Drug Administration approved the OraQuick OMT in 2004 for testing by health providers for individuals >12 years.We evaluated the diagnostic performance of OMT compared with routine BBT in children and adolescents aged 18 months to 18 years in Kenya and Zimbabwe.This analysis includes pooled data from 2 studies in Kenya and Zimbabwe that include parallel point-of-care diagnostic OMT and BBT to assess sensitivity and specificity of OMT among children and adolescents. Data were combined to increase precision of sensitivity and specificity estimates, as the number of newly diagnosed HIV-positive children in both settings has reduced with the scale-up of pediatric HIV prevention and treatment programs.This analysis was nested within the “Bridging the Gap in HIV Testing and Care for Children in Zimbabwe” (B-GAP Project) whose aim is to evaluate index-linked testing for pediatric case detection. Study participants were children and adolescents of unknown HIV status, aged 2–18 years, attending any health services in the participating hospitals and primary health care clinics.13) were recruited after determining HIV status using BBT within the trial. Second, children from outpatient clinics were recruited after HIV testing using BBT within routine testing; here children who tested BBT positive were oversampled.The “Saliva Testing and Video Information to Expand Uptake of Pediatric HIV Testing” (STEP-UP) study enrolled children aged 18 months to 12 years. Two recruitment streams were used. First, children of HIV-positive adults attending HIV clinics who were tested for HIV within a randomized controlled trial of financial incentives for index case testing  (fourth generation), followed by First Response  (third generation), if Determine was reactive. In the case of 2 reactive BBTs, the same 2 BBTs were performed by a different provider to confirm a positive diagnosis. In the case of discordant BBTs, both tests were repeated. If discordance persisted, a third test, CHEMBIO was performed . If this third test was positive, the result was reported as inconclusive and a retest conducted in 14 days. OMT was conducted by clinic staff blinded to BBT results.Testing followed the national algorithm16 In addition BBT was conducted by research staff for those enrolled in the FIT trial and non-research staff for those enrolled from routine testing points. Research staff performed OMT and were not blinded to BBT results.The national algorithm mirrored that in Zimbabwe with the following exceptions: the Determine HIV test was third instead of fourth generation and DNA PCR from dry blood spot specimens was the third test and was considered conclusive.The reference standard used for our study was the HIV status as per the national algorithim of each country.17 OMT results were read once between 20 and 40 minutes in Zimbabwe, and twice in Kenya at both 20 and 40 minutes to assess test performance at the lower and upper recommended times. OMT results were not shared with caregivers, because the test was undergoing validation.In Zimbabwe and Kenya, OMT sample collection and processing was performed bedside by qualified HIV testing lay providers who are typically lower than nurse level providers and are responsible for HIV testing in both countries. The qualification for these providers is a standard national training for HIV services conducted over 2 weeks. Testing was conducted according to manufacturer details , whereby the research staff collected an oral fluid sample from the participants by running the test device between the lips and outer gums of the client once on top and once at the bottom and then place the test device pad directly into the reaction fluid immediately after collection.Data were analyzed using STATA 14 . Sensitivity was calculated by dividing the number of OMT and BBT-positive children by the number of BBT-positive children. Specificity was calculated by dividing the number of OMT and BBT-negative children by the number of BBT-negative children. Positive predictive value (PPV) and negative predictive value (NPV) were calculated in the Zimbabwean cohort by dividing the number with both positive OMT and BBT by all the positive OMT tests (PPV) and by dividing the number with both negative OMT and BBT results by the total negative by OMT (NPV). PPV and NPV were not calculated in the Kenyan cohort, because positive children were oversampled. Ninety-five percent (95%) or 97.5% (when the estimate was 100%) confidence intervals (CIs) were calculated using a binomial distribution. Stability of the test results using result interpretation pictures from the manufacturer was described in Kenya.Adolescents ≥16 gave independent written informed consent without parental/guardian consent. Parents/guardians of children aged 18 months–15 years provided written consent; adolescents 13–15 years signed a paragraph within the parental consent form to give their assent, whereas children 7–12 years signed a separate assent document, which was optional in Kenya. B-GAP received approval from the Biomedical Research and Training Institute, the Medical Research Council of Zimbabwe and institutional review boards at Duke University and the London School of Hygiene and Tropical Medicine. The Kenyan study received approval from the Kenyatta National Hospital Ethics and Research Committee and the University of Washington Institutional Review Board.Overall, 1776 children were enrolled; 1570 (88%) from Zimbabwe and 206 (12%) from Kenya. The median age was 7.3 years (IQR: 4.7–11.6); 2 (0.1%) were 18 months −2 years; 512 (29%) were >2–5 years; 845 (48%) were >5–12 years; and 417 (23%) were >12–18 years. Overall, 918 (52%) were female. Among Kenyan children, 169 (82%) were identified via index case testing and 37 (18%) in outpatient clinics and inpatient wards  were positive by OMT. Among 1705 children negative by BBT, 1703/1705 (specificity: 99.9% [95% CI: 99.6% to 100.0%]) were negative by OMT. In the 1570 Zimbabwean participants, the PPV was 93.3% (95% CI: 77.9% to 99.2%) and the NPV was 100.0% (97.5% CI: 99.8% to 100.0%).In Zimbabwe, 2 children who initially tested BBT-negative and OMT-positive were retested within 1 week to confirm HIV status because of suggestive clinical presentation and history; both were confirmed positive. A 9-year-old was confirmed positive by ELISA. A 2-year-old was confirmed positive by First Response and CHEMBIO. Excluding these 2 children, the sensitivity and specificity of OMT compared with those of BBT were each 100%   had positive OMT at 40 minutes. Among the 163 children with negative OMT at 20 minutes, 163 (100%) had a negative OMT at 40 minutes. Using result interpretation pictures from the manufacturer, among 43 positive OMT results, 26 (60%) and 29 (67%) were strongly positive at 20 and 40 minutes, respectively. Three reads that were weakly positive at 20 minutes were strongly positive by 40 minutes; the remaining 14 (33%) were weakly positive at both 20 and 40 minutes.In this cross-sectional study of children aged 18 months to 18 years, we found that OMT had excellent sensitivity and specificity. When compared to the Kenyan and Zimbabwean national algorithms, OMT did not miss any positive children. These data suggest that OMT is valid for HIV diagnosis in this age range.18 In adults, antibody-based tests have limitations due to a long window period, which may lead to failure in detecting recent HIV infection.19 However, this is less of a concern among older children and younger adolescents who, if infected, are likely to have long-standing HIV, acquired perinatally.As with other antibody tests, OMT is inappropriate as a diagnostic test for children under 18 months due to the presence of maternal antibodies.26 Outpatient provider-initiated testing and counseling can identify children earlier in disease progression27; however, achieving high coverage is challenging21 because of high client volume and workload for limited numbers of HCWs.28 In resource-limited settings, scaling up testing will require simultaneously increasing coverage and minimizing costly components of testing, including HCW time.30 The ease and safety of OMT presents a potential opportunity for task-shifting from HCWs to lay providers, as was done in this study, or to caregivers to overcome human resource constraints. It is also important to note that the time to perform OMT is also similar to that required for BBT. Future research is needed to explore the acceptability and feasibility of OMT by caregivers and HCWs in facility and community settings.Our results provide evidence for wider use of OMT for pediatric testing. Current testing approaches to identify children include index-linked testing, provider-initiated testing and counseling, targeted testing in health facilities, and community-based testing.10 Despite this, the concentration of antibodies in oral fluid is lower than in blood and typically wanes during HIV treatment.32 Previous studies in Zimbabwe have confirmed that OMT has suboptimal sensitivity in ART-experienced children.33 WHO has issued warnings, advocating that rapid diagnostic tests not be used among ART-experienced adults; similar warnings seem warranted in children. Therefore, it is critical to avoid use of OMT by ART-experienced patients, either to confirm being “cured” of HIV or when reinitiating HIV care.34 Our study included an entirely ART-naive pediatric population and observed no false-negative results. In 2 cases, children were negative by BBT and positive by OMT and were confirmed HIV-positive upon repeat testing. This suggests slightly better detection by OMT than BBT in our study; it is unclear why we observed this counterintuitive finding.A 2012 systematic review comparing OMT with whole blood specimens reported a pooled sensitivity of 98.0% and specificity of 99.7% for OMT.31 An additional limitation is that in our study we did not have any inconclusive test results. Procedures on how to report and manage inconclusive test results must be put in place.Our study's strengths include a large sample of ART-naive, HIV-positive children to inform precise estimates of sensitivity. In addition, data from Kenya and Zimbabwe provided similar results. OMT results were compared with routine, field-based BBT according to national algorithms, which provides an apt comparison with standard-of-care tests and provides useful public health information. Limitations include that OMT result interpretation was not blinded in Kenya, which may have influenced result interpretation. National algorithms between the 2 countries differed slightly, so the “reference standard” was not the same in both countries. However, in both cases, the algorithms are those used for national guidelines. Consequently, our findings demonstrate the performance of OMT against the standard-of-care and are therefore generalizable in these settings. Although the BBT in this study was not ELISA or polymerase chain reaction (PCR) based, OMT has previously been compared with these more sensitive lab-based tests to inform Food and Drug Administration approval and WHO endorsement for use in adults.OMT is highly sensitive and specific in children and adolescents. This is consistent with findings from studies in adult populations. Policymakers and regulators should consider expanding the age at which OMT may be used to include children over 18 months.


The results indicate that the presence of PFR particles increased the storage modulus and decreased the impact energy determined by means of dynamic-mechanical-thermal analysis and falling weight impact tests respectively. Nevertheless, the reduction of impact energy was found to be lower in ABS/PFR samples than in neat ABS with increasing weight reduction. This effect was attributed to the lower cell sizes and higher cell densities of the microcellular core of ABS/PFR parts. All ABS/PFR foams showed a self-extinguishing behavior under UL-94 burning vertical tests, independently of the weight reduction. Gradual decreases of the second peak of heat release rate and time of combustion with similar intumescent effect were observed with increasing weight reduction under cone calorimeter tests.The present work deals with the study of phosphorus flame retardant microcellular acrylonitrile–butadiene–styrene (ABS) parts and the effects of weight reduction on the fire and mechanical performance. Phosphorus-based flame retardant additives (PFR), aluminum diethylphosphinate and ammonium polyphosphate, were used as a more environmentally friendly alternative to halogenated flame retardants. A 25 wt % of such PFR system was added to the polymer using a co-rotating twin-screw extruder. Subsequently, microcellular parts with 10, 15, and 20% of nominal weight reduction were prepared using a MuCell Acrylonitrile–butadiene–styrene (ABS) is one of the most used engineering polymers due its good combination of properties and low cost, being widely used in different industrial areas such as in the automotive sector, building, and construction, as well as in electrical and electronic applications. In the automobile sector, ABS is commonly used for interior and exterior car parts due to its high thermal insulation performance and the fact that its electrical properties do not change significantly with temperature and humidity . NeverthTraditionally, the flame retardancy enhancement of polymeric materials such as plastics, foams, resins, and adhesives has been achieved through the use of brominated flame retardant additives. These materials were introduced in the 1960s and 1970s and are very effective at low concentrations . HoweverAlthough styrenic polymers such as ABS are non-charring polymers, halogen-free phosphorous-based flame retardants  are still the most used alternative to halogen-based FRs . It is k® injection-molding physical foaming process or the supercritical gas dissolution batch foaming process 3Al, has a phosphorus content of 23.3–24.0 wt %, a density of 1.35 g/cm3 and an average particle size of 30 µm, as reported by the manufacturer.An acrylonitrile–butadiene–styrene copolymer (ABS), with the commercial name ELIXBefore compounding, the ABS pellets and the phosphorus flame retardants (PFR) powders were respectively pre-conditioned at 80 °C during 4h and at 100 °C during 12h.Neat ABS and ABS containing 12.5 wt % of APP and 12.5 wt % of AlPi  were melt-mixed in a co-rotating twin-screw extruder  at a constant rotating speed of 110 rpm and a temperature profile from entrance to die of 160–170–185–190–190 °C. At the end, the extrudates were water-cooled and pelletized. Prior to injection-molding, extruded pellets of neat ABS and ABS/PFR were dried at 80 °C for 4 h. This APP and AlPi ratio was previously studied in ABS, where the mechanisms and mode of action of APP/AlPi were discussed and related to the enhancement of the fire performance of ABS .® supercritical fluid (SCF) series II 25-mm injection valve, a SCF SII delivery system  for the conveying of SCF N2 and a mold temperature controlling device, were used for preparing the foamed parts. An injection temperature profile of 160–170–185–190–190 °C from hopper to nozzle was employed. The mold contained a 100 × 100 × 5 mm square-shaped plate cavity with a single fan gate located at one of the ends of the plate . The experimental conditions were a result of a prior optimization of the injection-molding foaming process in order to obtain the mentioned nominal weight reductions.A Victory 110 injection-molding machine  with a clamping force of 1100 kN, equipped with a 40 mm screw, a MuCelllate see . The N2 ϕ), cell nucleation density (N0) and cell density (fN) of the core of all microcellular foams were determined from the analysis of a minimum of five characteristic ×500 magnification SEM micrographs taken from the foamed core according to the intercept counting method [VDϕ, VD representing the vertical direction, i.e., the cell size in the thickness direction; and WDϕ (WD—width direction). On the other hand, N0 and fN were calculated assuming an isotropic distribution of spherical cells according to Equations (1) and (2)n is the number of cells in the micrograph, A (in cm2) its area, and rcρ is the relative density, determined as the quotient between the density determined at the core center of the foamed parts and the density of the unfoamed reference material; and in Equation (2) ϕ is the average cell size determined as the average of the measured cell sizes in VD and WD directions . In Equations (1) and (2), N0 represents the number of cells per volume of unfoamed material and fN the number of cells per volume of foamed material. Also, the relative density of the foamed parts, rρ, was determined as the quotient between their density and the density of the unfoamed reference material.The central zone of the parts was analyzed by means of scanning electron microscopy (SEM) using a JEOL JSM-5610 microscope. Samples were prepared by cryogenically fracturing the foams using liquid nitrogen and sputter depositing at their surfaces a thin layer of gold using a BAL-TEC SCD005 Sputter Coater. The values of the average cell size  was used to study possible differences in the storage modulus, loss modulus and tan δ of the unfoamed and foamed parts. A DMA Q800 from TA Instruments  was used and calibrated in a single cantilever configuration. The experiments were performed from −90 to 135 °C using liquid nitrogen at a constant heating rate of 2 °C/min and frequency of 1 Hz, applying a dynamic strain of 0.1%. Test specimens were cut from the center of the parts see  with a tFmax), energy absorbed until the maximum impact force (Emax) and total absorbed energy (ET) were registered.Impact tests were carried out on an instrumented vertical falling weight testing machine CEAST Dartvis  at room temperature using a drop mass of 27.96 kg and a drop height of 1 m, according to ISO 6603-2 standard. The injection-molded parts (100 × 100 × 5 mm) were freely supported on a steel ring of inner diameter 40 mm and a transverse collision between the hemispherical indentation tip (20 mm diameter) and the part was applied. The impact force was recorded as a function of time by means of a piezoelectric force transducer with a load cell of 4 kN mounted on the head indentation tip. The signal was processed by the CEAST DAS 16000 advanced data acquisition system with a frequency of 1 MHz. Three different samples were tested for both unfoamed and foamed parts. Values of the maximum impact force  ignited from bottom in the vertical configuration according to UL-94 standard .2 using a constant distance between the electrical resistance and the specimen of 25 mm. Heat release rate (HRR) vs. time curves were registered during the tests. Typical fire-reaction parameters such as time to ignition (TTI), peak of the heat release rate (PHRR) and total heat emitted (THE) were obtained from the cone calorimeter tests.Reaction-to-fire tests were carried out by means of a cone calorimeter  according to ISO 5660 standard procedure. Unfoamed and foamed specimens of 100 × 100 × 5 mm were irradiated with a constant heat flux of 50 kW/m® injection-molding foaming process showed a characteristic solid skin, a transition zone with decreasing density and a microcellular core structure. In proportion, the skins and transition zones represented around 30–40% of the whole thickness of the part and the microcellular core around 60–70%.First of all, as can be seen by the characteristic low magnification SEM images displayed in fN and N0 values clearly higher than 108 cells/cm3, in one case even surpassing 109 cells/cm3 (ABS/PFR-15). No clear relation was found between the weight reduction and the average cell size. For instance, ABS-15 foams presented lower average cell sizes than ABS-10 and ABS-20 foams, while ABS/PFR foams presented average cell sizes almost identical independently of the weight reduction (around 8–9 µm). Also, in terms of morphology all foams presented a homogeneous isotropic-like microcellular structure formed by spherical cells having aspect ratios (ϕVD/ϕWD) around 1.As can be seen from SEM images , clear dIn terms of PFR particles distribution, it can be seen by observing the characteristic SEM images presented in E′), loss modulus (E″) and tan δ were obtained from DMTA. E′ indicates the ability of a material to storage elastic deformation energy, while E″ describes the energy dissipation of a material when it is deformed, being a measurement of the energy loss [E″/E′). In the present work, the glass transition temperature (Tg) of the rubbery and rigid phases were determined using tan δ curves. It should be mentioned that there was a slight difference between the onset and the end of tan δ associated to the glass transition of SAN. The end of the transition was taken as reference to determine its intensity (peak of tan δ—end of tan δ).The storage modulus  as the nominal weight reduction increased. This fact indicates that the stiffness of ABS and ABS/PFR is affected by the foamed structure. However, ABS/PFR-20 foam showed an identical storage modulus to that of the unfoamed ABS, and ABS/PFR-10 and ABS/PFR-15 foams even higher.The values of Tg1) and that observed at higher temperature to the styrene-acrylonitrile (SAN) (Tg2) one [Tg2) one .Tg1 and Tg2) and that the foamed parts globally displayed slightly lower values when compared to the respective unfoamed counterparts, indicating that the presence of the PFR particles and/or the microcellular structure of the foamed parts contributed to slightly decrease the chemical interactions between ABS’ macromolecules. The highest observed reduction was of 3.7 °C between the Tg2 of the unfoamed ABS and ABS/PFR-10 foam.Also, from Moreover, a chain mobility reduction was observed during the glass transitions of the rubbery and rigid phases with the presence of APP and AlPi particles a,b, resuFmax than ABS and ABS/PFR foamed parts, as well as higher Emax and ET. As can be seen in ET and Emax, much higher than in the case of ABS/PFR materials, which was related to a more significant plastic deformation during impact. Researchers have argued that the impact resistance reduction in cellular parts is due to the stress concentrator role of cells [Unfoamed parts displayed higher values of of cells . This imof cells .Fmax than pure ABS, as well as a totally brittle fracture. This behavior has also been observed in polymeric compounds with phosphorous-flame retardant additives [Fmax, Emax, and ET , while all ABS/PFR samples showed a self-extinguishing behavior (UL-94 V0 classification). This self-extinguishing behavior was associated to the flame inhibition promoted by the liberation of phosphorus radicals during the hydrolysis of AlPi and the formation of an effective protective layer due to the strong interactions between PFR particles and the polymer, being APP the major char-promoting component .The forced flaming fire behavior was assessed by means of cone calorimeter tests. 1). On the contrary, ABS/PFR showed a typical curve of a thermal thick charring material with an additional peak at the end of burning [As can be seen, the burning behavior of ABS and ABS/PFR gave rise to different characteristic HRR versus time curves. ABS showed a typical curve of a thermal intermediate thick non-charring and no residue forming material , a gradual reduction of the second PHRR (PHRR2) and time of combustion (see tion see  was obseMoreover, ABS/PFR foams showed similar residue contents see  and resiThe morphology of the char residue was also analyzed to assess the structure formed during combustion. All ABS/PFR parts, independently of the weight reduction, showed a porous carbonaceous structure see , which lMicrocellular ABS and ABS/PFR parts with 10, 15, and 20% of weight reduction were prepared and characterized. They presented a cellular structure gradient with skin, transition zone of decreasing density and a microcellular core. The addition of APP and AlPi into ABS promoted the formation of a more homogeneous microcellular core structure with smaller cell sizes and higher cell densities than neat ABS.Both unfoamed and foamed ABS/PFR parts globally showed an increased storage modulus and a slight decrease of the butadiene and SAN glass transition temperatures compared to neat ABS. The incorporation of the PFR system and the foaming process led to a change in the ABS fracture behavior from ductile to brittle. Nevertheless, the reduction in impact energy was found to be lower in ABS/PFR parts than in neat ABS with increasing weight reduction.Compared with the unfilled ABS, unfoamed and foamed ABS/PFR displayed an improved fire behavior. Both unfoamed and foamed ABS/PFR parts showed a UL-94 V0 classification and similar intumescent effect under the cone calorimeter tests. Compared with the unfoamed ABS/PFR, foamed ABS/PFR parts showed a gradual reduction of the second PHRR and time of combustion with increasing weight reduction.