Case Series of Endoscopic Papillary Balloon Dilation for Children with Common Bile Duct Stones and a Review of the Literature.

Background: Endoscopic sphincterotomy (EST) has been employed for the endoscopic treatment of common bile duct stones (CBDSs) and has been reported to have a high success rate for stone removal. However, EST is associated with a risk of bleeding, perforation, and sphincter of Oddi function disruption. To avoid these risks, endoscopic papillary balloon dilation (EPBD) is an option for CBDS. Sphincter of Oddi function preservation decreases long-term biliary infection and gallstone recurrence. EPBD may have advantages in children who require a long follow up. However, there have been few reports on pediatric cases, particularly in infants. Methods: From September 2017 to December 2023, we performed EPBD for four pediatric CBDSs. The patients were aged from 5 months to 8 years, including two infants aged 5 and 6 months. Furthermore, we reviewed the stone removal rate and complications of 545 ESTs performed at high-volume centers and 13 EPBD-reported cases in children with CBDSs. Results: CBDSs of all patients who underwent EPBD in our institution were successfully removed. No bleeding or perforation was noted; pancreatitis was observed in three patients. In an analysis of 545 ESTs in children, the stone removal rate was high, ranging from 83% to 100% (mean 96%). The incidence of pancreatitis was 0-9.6% (mean 4.4%), and the grade of pancreatitis was almost mild. The bleeding frequency was 1.3-5.4% (mean 2.7%). With regards to the grade of bleeding, seven cases were mild (64%) and four were moderate (36%). Compared with adults who underwent EST, the frequencies of pancreatitis and bleeding were almost equal in children; however, in children, once bleeding occurs, it has a higher risk of leading to blood transfusion. Stone removal via EPBD in children has a 100% success rate. Pancreatitis was responsible for all complications were related; its frequency was 46% (6/13 patients, including five mild cases and one moderate case), which is higher than that of EST and adult cases who underwent EPBD. In most children with pancreatitis, pancreatic enzyme levels returned to normal within 2-3 days following EPBD, and no severe cases caused by EPBD were reported. Conclusions: CBDS removal via EPBD in children has a high success rate with very low risk of bleeding and perforation. Although pancreatitis frequently occurs, most cases are mild. Sphincter of Oddi function preservation via EPBD is expected to prevent long-term stone recurrence and biliary tract infection, and EPBD is considered to be an effective method for CBDS removal in children.

Development of a Microminipig Model of Atherosclerosis for the Evaluation of a HMGCR Inhibitor.

BACKGROUND/AIM: Atherosclerosis is known as a major risk factor for cardiovascular disease, and development of an animal model of atherosclerosis is required to investigate its clinical pathogenesis. We studied the optimal amount of cholesterol in the diet and the optimal experimental period for development of a Microminipig model of atherosclerosis for the evaluation of a hydroxymethylglutaryl-CoA reductase (HMGCR) inhibitor (atorvastatin). MATERIALS AND METHODS: Eighteen male animals (3-4 months old) were divided into 3 groups. Group 1 consisted of control animals receiving a normal chow diet, Group 2 animals received a high fat (12% w/w) and low cholesterol (0.1% w/w) diet (HFLCD), and Group 3 animals received HFLCD+statin for 12 weeks. Animals received statin at 3 mg/kg body weight per day. HFLCD did not down-regulate the hepatic expression of HMGCR mRNA. RESULTS: HFLCD increased body, omentum, and mesenteric adipose tissue weight, and induced hypercholesterolemia and atherosclerotic lesions in the abdominal aorta. HFLCD+statin inhibited hypercholesterolemia and atherosclerotic lesions, but not obesity. CONCLUSION: A microminipig atherosclerosis model induced by HFLCD can be used in the evaluation of HMGCR inhibitors for the treatment of hypercholesterolemia and atherosclerosis.

Mini-probe endoscopic ultrasound for the diagnosis of congenital esophageal or duodenal stenosis.

PURPOSE: The usefulness of endoscopic ultrasound (EUS) in pediatric populations has been recently appreciated; however, published studies on mini-probe EUS in the diagnosis of congenital esophageal stenosis (CES) or congenital duodenal stenosis (CDS) in pre-school patients remain scarce. This study aimed to report the utility of mini-probe EUS for the diagnosis of CES or CDS in pre-school patients based on the etiology. METHODS: We retrospectively reviewed the medical records of pediatric patients with CES or CDS who underwent mini-probe EUS through the stenotic segments at our hospital between December 2006 and December 2021. RESULTS: Five patients with CES and one with CDS were enrolled. The median age and body weight when EUS was performed were 12.5 months and 8.5 kg, respectively. Hypoechoic lesions were observed on EUS in three patients, which were assessed as cartilage; one patient had no hypoechoic lesion but had a focal thickness of the muscular layer. They were diagnosed with tracheobronchial remnants based on EUS. The full circumferential wall thickness of the esophagus was visualized in one patient with fibromuscular hypertrophy. The histopathological findings confirmed the diagnoses. In the patient with CDS, EUS findings revealed pancreatic parenchyma encircling the stenotic part of the duodenum. The preoperative diagnosis was annular pancreas. The patient underwent duodenoduodenostomy, and intraoperative findings confirmed the diagnosis. CONCLUSION: Mini-probe EUS can be recommended as a feasible and safe technique for infants and toddlers. It can effectively diagnose CES or CDS based on etiology and can inform treatment strategies for pre-school patients.

Cyanobacterial photosystem II reaction center design in tobacco chloroplasts increases biomass in low light.

The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 µE m-2 s-1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.

Recurrent massive bleeding from a small intestinal arteriovenous malformation after surgery for biliary atresia in an infant: a case report and literature review.

BACKGROUND: Small intestinal arteriovenous malformation (AVM) can cause bleeding. Most small intestinal AVMs occur during adulthood, rarely in infancy. We report a case of an infant with hemorrhage due to small intestinal AVM early and recurrently after Kasai portoenterostomy (PE) for biliary atresia (BA). CASE PRESENTATION: A 51-day-old male infant was admitted to our institution for obstructive jaundice. Laparotomic cholangiography revealed BA (IIIb1µ), and Kasai PE was performed at 60 days of age. On postoperative day 17, he developed massive melena and severe anemia. Contrast-enhanced computed tomography (CT) revealed that the jejunum around the PE site was strongly enhanced with enhancing nodules in the arterial phase, and a wide area of the Roux limb wall was slightly enhanced in the venous phase. As melena continued, emergency laparotomy was performed. There were no abnormal macroscopic findings at the PE site except for a clot in the Roux limb 5 cm away from the PE site, and the Roux limb was resected 5 cm. On further investigation, a red spot was detected on the jejunal serosa 30 cm away from the Roux-en-Y anastomosis site. PE and wedge resection for the red spot were performed. Histopathologically, both specimens indicated AVM. He was jaundice-free 65 days after the first surgery. However, at 7 months of age, he developed massive melena again. Contrast-enhanced CT and upper gastrointestinal endoscopy revealed no bleeding lesions. Hemorrhagic scintigraphy showed a slight accumulation at the hepatic hilum prompting an emergency surgery. Intraoperative endoscopy detected a bleeding lesion at the PE site, and the Roux limb was resected (approximately 6 cm). Intraoperative frozen section analysis of the stump of the resected jejunum revealed no abnormal vessels. PE was performed, and permanent section analysis revealed an AVM in the resected jejunum. The postoperative course was uneventful without re-bleeding. CONCLUSIONS: We experienced a case of recurrent massive bleeding from small intestinal AVM in an infant after surgery for BA. Intraoperative endoscopy and frozen section analysis helped identify the bleeding lesion and perform a complete resection of the small intestinal AVM, even after surgery, in the infant.

Current status and future challenges in the endoscopic management of non-variceal upper gastrointestinal bleeding in children.

PURPOSE: This study aimed to elucidate the characteristics of non-variceal upper gastrointestinal bleeding (NVUGIB) in children and the outcomes of endoscopic hemostasis (EH) performed by pediatric surgeons. METHODS: We retrospectively reviewed the medical records of pediatric patients with NVUGIB who had undergone esophagogastroduodenoscopy (EGD) at our hospital, between December 2006 and March 2020. RESULTS: Thirty-six patients were included. The most common endoscopic diagnosis was gastritis (28%), followed by gastric ulcer (25%) and duodenal ulcer (25%). Thirty patients (83%) had underlying diseases, including a recent viral infection (39%), followed by Helicobacter pylori infection (14%). Fourteen patients (39%) underwent EH, exhibiting a high initial rate of hemostasis (100%). This was achieved in 12/14 patients by clipping. One patient was treated using a combination of a hypersaline and epinephrine (HSE) injection and coagulation therapy, as the ulcer exceeded the width of the hemoclips. Rebleeding occurred in one patient (7.1%) initially treated with pure ethanol injections. The partial view of the pediatric duodenum prevented the placement of hemoclips; hence, the patient was treated using a combination of HSE and coagulation therapy. CONCLUSION: Clipping, injection, and/or coagulation therapy can lead to high success rates for hemostasis, considering the anatomical characteristics in a pediatric population.

Safety and efficacy of mucous fistula refeeding in low-birth-weight infants with enterostomies.

PURPOSE: To investigate the safety and efficacy of mucous fistula refeeding (MFR) in low-birth-weight infants. METHODS: Between December 2006 and December 2018, medical records of low-birth-weight infants who underwent small bowel enterostomy formation in the neonatal period and subsequent stoma closure at our institution were retrospectively reviewed. Patients were assigned to "refeeding" (RF) and "non-refeeding" (NRF) groups, which were compared for patient characteristics and clinical outcomes. We also cultured the proximal stoma output over time in the RF group and reviewed changes in the flora to evaluate the safety of refeeding. RESULTS: In the RF group, compared with that before refeeding, there was significantly more rapid weight gain after refeeding (17.7 vs 10.6 g/day; P = 0.002). Median total time of parenteral nutrition (PN) was 25 and 87 days in the RF and NRF groups, respectively (P = 0.001). The number of patients who developed PN-associated liver disease (PNALD) was smaller in the RF group (P = 0.12). No complications of MFR were noted and no pathogenic bacteria were cultured. CONCLUSION: MFR was able to diminish the need for PN, which potentially decreased the incidence of PNALD, and was safe as there were no complications of the refeeding process.

Recurrent intestinal ischemia following surgery for gastric and duodenal perforations: a case report.

BACKGROUND: Extremely low birth weight (ELBW) is a risk factor for various gastrointestinal complications. In the recent decades, advances in medicine have increased the survival of ELBW infants with necrotizing enterocolitis (NEC). To our knowledge, there have been no reports of neonates or infants developing simultaneous gastric and duodenal perforations and later developing NEC. We report a case of an extremely low birth weight infant (ELBWI) who developed both gastric and duodenal perforations at the same time and developed NEC after operation for gastric and duodenal perforations. CASE PRESENTATION: The patient was a female infant with ELBW who developed both gastric and duodenal perforations at the same time and developed NEC after operation for gastric and duodenal perforations. After birth, endotracheal intubation was performed. However, barotrauma occurred during positive pressure ventilation, resulting in a large area of emphysema in the left lower lung field, leading to collapse of the left lung. This giant bulla may have compressed the pulmonary vein, possibly resulting in pulmonary venous thrombosis (PVT). This episode could have triggered simultaneous gastric and duodenal perforations. In addition, we hypothesized that PVT caused acute arterial ischemia, leading to the development of NEC. The infant was started on heparin for anticoagulation. Later, the infant developed non-immunoglobulin E (IgE)-mediated gastrointestinal food allergies (non-IgE-GI-FAs). The giant bulla associated with barotrauma might have caused PVT, resulting in arterial emboli and multiple simultaneous gastrointestinal perforations. CONCLUSIONS: Anticoagulation therapy with heparin for acute arterial thrombosis is effective for preventing the development of short bowel syndrome. Duodenal and intestinal surgery probably acted as risk factors for the subsequent development of non-IgE-GI-FAs. The infant had been stabilized at the time of writing this report.

Neonates undergoing gastrointestinal surgery have a higher incidence of non-IgE-mediated gastrointestinal food allergies.

PURPOSE: We aimed at investigating the incidence and risk factors of non-IgE-mediated gastrointestinal food allergies (non-IgE-GI-FAs) in neonates and infants. METHODS: A total of 126 infants who underwent neonatal gastrointestinal surgeries were grouped into those with (n = 13) and those without an onset of non-IgE-GI-FAs (n = 113). The characteristics of the two groups (e.g., birth weight, delivery type, small intestinal surgeries, and pre-/postoperative nutrition) were compared. Small intestinal surgeries were classified into those with and those without full-layer invasion of the small intestine. For the statistical analysis, postoperative nutrition was classified into breast milk only, formula milk, and elemental diet only. RESULTS: Except for full-layer surgical invasion of the small intestine and the period of parenteral nutrition, no significant differences were found between the two groups. Surgery with full-layer invasion was a risk factor of non-IgE-GI-FAs (odds ratio (OR) 10.70, 95% confidence interval (95% CI) 2.11-54.20; p = 0.004). Formula milk after surgery was a risk factor of non-IgE-GI-FAs when compared to breast milk (OR 5.65, 95% CI 1.33-24.00; p = 0.019). CONCLUSION: Neonates undergoing gastrointestinal surgery have a higher incidence of non-IgE mediated gastrointestinal food allergies. We recommend that formula milk should not be administered to newborns who underwent neonatal gastrointestinal surgeries with full-layer invasion of the small intestine.

Efficacy of an anal fistula plug for fistulas-in-Ano in children.

BACKGROUND: In children, perianal abscesses have a good prognosis and often heal with age. However, some perianal abscesses are refractory to treatment and remain as fistulas-in-Ano. Treatment with a Surgisis Anal Fistula Plug® has been reported as a new method of treatment for fistulas. In adults, the plug has been reported to cause little pain and have a high cure rate, but there have been no reported cases of its use in children. PURPOSE: This study was designed to analyze the efficacy of the plug for closure of refractory fistulas in children. METHODS: Since the plug has not been approved as a medical device in Japan, application for its use was submitted to the ethics committee of our university, and approval was granted, marking the first use of the plug in Japan. We classified refractory fistulas as those treated for 6months or longer and remaining unhealed, even after 1year of age, despite continued conservative treatment. The plug was used in 11 refractory fistulas in 8 children. RESULTS: Eight of 11 fistulas (72.7%) were successfully treated. Three fistulas recurred, and fistulectomies were performed. No sequelae were observed after AFP treatment. CONCLUSION: The plug was effective even for closure of refractory fistulas without sequelae in children. LEVELS OF EVIDENCE: Treatment Study, Level IV.

Why p-Cymene? Conformational effect in asymmetric hydrogenation of aromatic ketones with a η(6) -arene/ruthenium(II) catalyst.

The global reaction route mapping (GRRM) methods conveniently define transition states in asymmetric hydrogenation and transfer hydrogenation of aromatic ketones via the [RuH{(S,S)-TsNCH(C6 H5 )CH(C6 H5 )NH2 }(η(6) -p-cymene)] intermediate. Multiple electrostatic CH/π interactions are the common motif in the preferred diastereometric structures.

A novel method of transgene delivery into triticale plants using the Agrobacterium transferred DNA-derived nano-complex.

Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particle-mediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.

Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation.

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations.

Prostaglandin F2α (PGF2α) stimulates PTGS2 expression and PGF2α synthesis through NFKB activation via reactive oxygen species in the corpus luteum of pseudopregnant rats.

This study was undertaken to investigate how prostaglandin F(2α) (PGF(2α)) increases PGF(2α) synthesis and PTGS2 expression in the corpus luteum of pseudopregnant rats. We further investigated the molecular mechanism by which PGF(2α) stimulates PTGS2 expression. PGF(2α) (3 mg/kg) or phosphate buffer as a control was injected s.c. on day 7 of pseudopregnancy. Ptgs2 mRNA expression and PGF(2α) concentrations in the corpus luteum were measured at 2, 6, and 24 h after PGF(2α) injection. PGF(2α) significantly increased Ptgs2 mRNA expression at 2 h and luteal PGF(2α) concentrations at 24 h. PGF(2α) significantly decreased serum progesterone levels at all of the times studied. Simultaneous administration of a selective PTGS2 inhibitor (NS-398, 10 mg/kg) completely abolished the increase in luteal PGF(2α) concentrations induced by PGF(2α). PGF(2α) increased NFKB p65 protein expression in the nucleus of luteal cells 30 min after PGF(2α) injection, and electrophoretic mobility shift assay revealed that PGF(2α) increased binding activities of NFKB to the NFKB consensus sequence of the Ptgs2 gene promoter. Simultaneous administration of both superoxide dismutase and catalase to scavenge reactive oxygen species (ROS) inhibited the increases of nuclear NFKB p65 protein expression, lipid peroxide levels, and Ptgs2 mRNA expression induced by PGF(2α). In conclusion, PGF(2α) stimulates Ptgs2 mRNA expression and PGF(2α) synthesis through NFKB activation via ROS in the corpus luteum of pseudopregnant rats.

Progesterone increases manganese superoxide dismutase expression via a cAMP-dependent signaling mediated by noncanonical Wnt5a pathway in human endometrial stromal cells.

CONTEXT: Manganese superoxide dismutase (Mn-SOD), an antioxidant enzyme in the mitochondria, protects cells by scavenging superoxide radicals in human endometrial stromal cells (ESCs). Mn-SOD increases in ESCs during decidualization induced by progesterone. OBJECTIVE: The present study investigated the molecular mechanism for Mn-SOD expression induced by progesterone in human ESCs. METHODS: ESCs were incubated with medroxyprogesterone acetate (MPA; 10(-6) m) or dibutyryl-cAMP (0.5 mm) for 17 d. To determine whether a cAMP-dependent signaling pathway is involved in the MPA-induced Mn-SOD expression, ESCs were treated with H89, an inhibitor of cAMP-dependent protein kinase A. A chromatin immunoprecipitation assay was performed to examine the binding of cAMP-binding protein to the cAMP-response element on the Mn-SOD gene promoter. To examine the involvement of Wnt5a signaling, anti-Wnt5a antibodies were used to neutralize the Wnt5a activities. RESULTS: Mn-SOD and Wnt5a mRNA levels and intracellular cAMP concentrations were significantly increased by MPA. These increases were accompanied by an increase in the mRNA expression of IGF-binding protein-1, a marker of decidualization. The increase in Mn-SOD mRNA levels by MPA or dibutyryl-cAMP was completely inhibited by H89. The chromatin immunoprecipitation assay revealed that MPA induced cAMP-binding protein binding with cAMP-response element on the Mn-SOD gene promoter. The increase in intracellular cAMP concentrations by MPA was completely inhibited by treatment with anti-Wnt5a antibodies. MPA treatment had no effects on ß-catenin expression. CONCLUSIONS: Progesterone increased Mn-SOD expression via a cAMP-dependent pathway in ESCs during decidualization. cAMP-dependent signaling stimulated by progesterone is mediated by noncanonical Wnt5a pathways that signal independently of ß-catenin.

Chiral eta(6)-arene/N-tosylethylenediamine-ruthenium(II) complexes: solution behavior and catalytic activity for asymmetric hydrogenation.

Aromatic ketones are enantioseletively hydrogenated in alcohols containing [RuX{(S,S)-Tsdpen}(eta(6)-p-cymene)] (Tsdpen=TsNCH(C(6)H(5))CH(C(6)H(5))NH(2); X=TfO, Cl) as precatalysts. The corresponding Ru hydride (X=H) acts as a reducing species. The solution structures and complete spectral assignment of these complexes have been determined using 2D NMR ((1)H-(1)H DQF-COSY, (1)H-(13)C HMQC, (1)H-(15)N HSQC, and (1)H-(19)F HOESY). Depending on the nature of the solvents and conditions, the precatalysts exist as a covalently bound complex, tight ion pair of [Ru(+)(Tsdpen)(cymene)] and X(-), solvent-separated ion pair, or discrete free ions. Solvent effects on the NH(2) chemical shifts of the Ru complexes and the hydrodynamic radius and volume of the Ru(+) and TfO(-) ions elucidate the process of precatalyst activation for hydrogenation. Most notably, the Ru triflate possessing a high ionizability, substantiated by cyclic voltammetry, exists in alcoholic solvents largely as a solvent-separated ion pair and/or free ions. Accordingly, its diffusion-derived data in CD(3)OD reflect the independent motion of [Ru(+)(Tsdpen)(cymene)] and TfO(-). In CDCl(3), the complex largely retains the covalent structure showing similar diffusion data for the cation and anion. The Ru triflate and chloride show similar but distinct solution behavior in various solvents. Conductivity measurements and catalytic behavior demonstrate that both complexes ionize in CH(3)OH to generate a common [Ru(+)(Tsdpen)(cymene)] and X(-), although the extent is significantly greater for X=TfO(-). The activation of [RuX(Tsdpen)(cymene)] during catalytic hydrogenation in alcoholic solvent occurs by simple ionization to generate [Ru(+)(Tsdpen)(cymene)]. The catalytic activity is thus significantly influenced by the reaction conditions.

High frequency Agrobacterium tumefaciens-mediated plant transformation induced by ammonium nitrate.

Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation efficiency. Here, we report that a threefold increase in the concentration of NH(4)NO(3) in the growth medium results in more than a threefold increase in the Agrobacterium tumefaciens-mediated transformation frequency of Nicotiana tabacum plants. Regeneration of calli without selection showed that the increase in transformation frequency was primarily due to the increase in transgene integration efficiency rather than in tissue regeneration efficiency. PCR analysis of insertion sites showed a decrease in the frequency of truncations of the T-DNA right border and an increase on the left border. We hypothesize that exposure to ammonium nitrate modifies the activity of host factors leading to higher frequency of transgene integrations and possibly to the shift in the mechanism of transgene integrations.