Oligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition.WES) to identify the cause of oligodontia in a 9‐year‐old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype.We applied whole‐exome sequencing (WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression.We identified a novel compound heterozygous missense mutation in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.Our results reveal a novel compound heterozygous variant in  Oligodontia is the severe form of tooth agenesis characterized by the developmental absence of six or more permanent teeth, not including the third molars        . Written informed consent was obtained from all study participants and their legal guardians in the case of minors.Subjects were recruited at the UTHealth School of Dentistry clinics, based on clinical and radiographic examination findings showing the absence of one or more permanent teeth (excluding third molars), supporting the diagnosis of tooth agenesis. Peripheral blood samples were collected as source of genomic DNA. DNA extraction followed established protocols and the DNA concentration and quality were estimated using NanoDrop ND‐1000 .The discovery family consisted of a nuclear family of a 9‐year‐old Asian girl with oligodontia and her parents. Examination of the child revealed absence of 11 permanent teeth   followed by massively parallel sequencing on an Ilumina HiSeq 2000 platform . Paired‐end reads were aligned against the human reference genome (UCSC Human Genome Browser hg19) using the Burrows‐Wheeler Aligner  (Li and Durbin Whole‐exome sequencing was performed on DNA samples of the child proband (II.1) after Sanger sequencing excluded mutations in www.evs.gs.washington.edu), or in the 1000 Genomes Project Database (www.1000genomes.org), (2) synonymous variants, and (3) intronic variants >5 bp from exon boundaries. Variants were classified as deleterious , benign, or of unknown clinical significance, by the algorithms SIFT, PolyPhen‐2, Mutation Taster, Mutation Assessor (implemented in the dbNSFP database), and VIPUR  (Table To further determine the frequency of the A) Table  in an adWNT10A (NM_025216.2) to other species was assessed using ClustralW2 . Protein sequence accession numbers were as follows: mouse (Mus), NP_033544.1; rat (Rattus), NP_001101697.1; human (Homo), NP_079492.2; chimp (Pan), XP_516098.2; monkey (Macaca), XP_001095740.1; wolf (Canis), XP_545648.2; chicken , NP_001006590.1; zebrafish, (Danio) NP_571055.1; and frog (Xenopus), XP_002934004.2  and 37% sequence identity to the template structure.Sequence homology of human 4.2 Fig. . Assessm4.2 Fig. , with a Danio rerio) wild‐type Dz and AB strains were maintained at 28.5°C, and embryos were collected and staged according to established methods . Two zebrafish wnt10a antisense morpholinos (MO) and one mismatch control MO were designed by GeneTools : (1) translation blocking MO (tbMO) targeting the ATG site in exon 1 (GTCGTGAGAGCTCATTCATGGAATC); (2) splice‐blocking MO (sbMO) targeting the exon2/intron2 junction (CTGTTTGATTTGATCGCTTACCCCT); and (3) mismatch control MO (GTCaTaAGAaCTCATTgATaGAATC) 2, 5.0 mmol/L HEPES pH 7.6) and 0.5 uL of 2% phenol red was added to facilitate injections. One‐cell embryos were injected with 1 nL of MO and observed during development up to 8 days postfertilization (dpf). Injection volume was calculated by measuring the diameter of injected droplet. Embryos were incubated at 28°C and collected at appropriate time points.Reciprocal BLAST was used against the TC) Fig. . All MOswnt10a was made by insertion of a reverse T7 promoter in pCS2 + Wnt10a plasmid using the primers . Probe was transcribed using digoxigenin‐11‐UTP  and T7 polymerase . Whole‐mount in situ hybridization was carried out as described . Following destaining in 3% H2O2/0.5% KOH for 10 min at room temperature, embryos were stained in 0.02% alizarin red solution containing 100 mmol/L Tris, pH 7.5, 25% glycerol for 30 min at room temperature. Embryos were destained in 50% glycerol/0.1% KOH for 30 min and stored in 50% glycerol. Imaging was performed using the LAS Montage Module .In order to visualize cartilage and bone structures during zebrafish embryo development, alcian blue  and alizarin red  staining was performed using standard techniques   and then transferred onto PVDF membrane . The membrane was incubated with primary antibodies anti‐wnt10a  and antiactin  at room temperature for 2 h. Following washing three times with 1xTBST for 15 min each, the membrane was incubated with IRDye® 800 CW antirabbit IgG secondary antibody  for 1 h at room temperature. Protein bands were visualized using LI‐COR Odyssey Scanning Imager (LI‐COR).Cell lysates were prepared from 4 hpf and 24 hpf UIC, wnt10a TbMO‐injected embryos, wnt10a mismatch control (MM)‐MO‐injected embryos, and wnt10a RNA‐injected embryos were collected at different time points  and homogenized in Trizol reagent (Invitrogen). Total RNA was isolated using RNeasy Mini kit , and treated with DNase I. Complementary DNAs (cDNA) were synthesized from 1 µg of total RNA using the iScript cDNA synthesis kit  according to manufacturer's instructions. Expression levels of wnt10a (ENSDARG00000017155), msx1 (ENSDARG0000000 7641), eda (ENSDARG00000074591), dlx2b (ENSDARG 00000017174), and axin2 (ENSDARG00000100149) mRNA were evaluated by qRT‐PCR using SYBR® Green PCR Master Mix (Applied Biosystems) in a Viia7 sequence detection instrument (Applied Biosystems). Assays were conducted in triplicates. Quantitative expression values were extrapolated from separate standard curves, and normalized to β‐actin as internal control. Fold‐changes were calculated by the 2−∆∆Ct method . The Forward (Fwd) primer contains partial morpholino target site . Four‐point synonymous mutations (underlined) were introduced in the morpholino target site to create mismatches to the morpholino in order to prevent morpholino binding. PCR products digested with EcoRI were then cloned using T4 DNA ligase into pCS2 vector, which had been digested with EcoRI and treated with alkaline phosphatase (Sigma) prior to ligation. One Shot TOP10 competent cells (Invitrogen) were used for transformation of the ligation reaction. pCS2‐wnt10a plasmid DNA was prepared using Qiagen plasmid mini kit (Qiagen). Clones with correct wnt10a full‐length sequence (NM_130980) were identified through DNA sequencing. Human full‐length WNT10A‐expressing vector pCS2‐WNT10a (NM_025216) was purchased from GeneScript.Total mRNA was isolated from zebrafish embryos collected at 24‐h postfertilization (hpf). Total mRNA (1 µg) was used as template to generate a cDNA library using Bio‐Rad iScript cDNA synthesis Kit (Bio‐Rad). Zebrafish full‐length wnt10a was amplified from the cDNA library using primers with a EcoRI restriction enzyme  cut site added at both ends for cloning purposes (Fwd: AAGGTTCCGAATTCDanio rerio (Dr) wnt10a and Homo sapiens (Hs) WNT10A were created using the QuikChange XL Site‐Directed Mutagenesis Kit . Primer sequences are available in Table μg/mL and injected 1 nL/embryo.Mutated versions of WNT10A as potentially pathogenic: c.637G>A (p.Gly213Ser) and c.1070C>T (p.Thr357Ile), located in exons 3 and 4 of the WNT10A gene, respectively. Sanger sequencing of exon/intron boundaries confirmed the presence of both variants in the affected child, whereas unaffected parents were found to be carriers for each variant (c.1070C>T in the father and c.637G>A in the mother) , p.Arg360Cys (0.96 score), and p.Phe228Ile (0.91 score)  and c.1070C>T (p.Thr357Ile) affect highly conserved amino acids and are predicted as deleterious  Table , and areThe known heterozygous missense variant c.682T>A (p.Phe228Ile) was identified in one patient with oligodontia of the 97 cases with tooth agenesis. None of the tested variants were found in the unaffected individuals.wnt10a during craniofacial and tooth development. We first assessed its expression in wild‐type zebrafish embryo heads by real‐time PCR. wnt10a expression was detected throughout developmental stages preceding and following critical time points for tooth development , although no significant differences were observed between each stage  or splice‐blocking (sbMO) morpholino . At 2 dpf, a significant reduction in the expression of dlx2b, eda, and axin2 (P = 0.001) was observed in wnt10a MO‐injected embryos. In contrast, overexpression of wnt10a resulted in increased expression of all four genes, with significant differences observed for msx1 (P = 0.01) and axin2 (P = 0.02). Injection of mRNA encoding the wnt10a mutant alleles also perturbed expression of msx1, dlx2b, eda, and axin2, particularly the G248S mutant (0.002 < P < 0.02) ; c.1070C>T (p.Thr357Ile) as likely pathogenic for isolated oligodontia, and demonstrate that perturbations in wnt10a expression impairs normal tooth development in zebrafish embryos.Numerous studies have implicated a major role for WNT10A gene is a member of the WNT gene family, which consists of a large family of secreted glycoproteins that specify various cell lineages during embryogenesis  identified here are rare and predicted to affect highly conserved amino acids with deleterious effects to the WNT10A protein structure and function. Furthermore, each mutation is predicted to be individually disruptive of protein function with potential additive effects. For example, these variants are likely destabilizing, or may disrupt post‐translational modifications and inhibit proper localization, or eliminate disulfide bonds, which are sufficient to cause loss of molecular function. Wnt proteins are highly conserved in vertebrates and have a remarkably high number of disulfide bonds, suggesting that protein conformation is important for correct interaction with their receptors. This is important because WNT10A likely requires the proper orientation of both binding regions to interact with its receptor(s) , while no WNT10A variant was found in unaffected individuals.Compound homozygous and heterozygous, and single heterozygous mutations in Wnt10a in a mouse model remains questionable and fails to elucidate the role of this gene in tooth agenesis. Evidence supporting a role for Wnt10a in the developing mouse tooth shows its expression in the enamel knot, and then shifting from the secondary enamel knot to the underlying mesenchyme to be continuously expressed in the developing odontoblasts  in fully differentiated secretory odontoblasts, indicating that Wnt10a is potentially involved in dentin mineralization events as an upstream regulator of Dspp  and molar teeth characterized by taurodontism and altered molar crown morphology , implicating a role for wnt10a during tooth development in zebrafish embryos.To gain better insights into the role of wnt10a knockdown arrests or severely impairs tooth development in almost all of the embryos with no additional gross structural abnormalities. Furthermore, perturbations of wnt10a expression resulted in altered expression of additional tooth development genes such as msx1, dlx2b, eda, and axin2 (Borday‐Birraux et al. wnt10a expression caused a significant reduction in the expression of msx1, dlx2b, eda, and axin2, thus suggesting that imbalances in wnt10a expression may directly or indirectly contribute to arrest or impairment of tooth development in zebrafish. In contrast, overexpression of wnt10a resulted in an increase in the expression of msx1, dlx2b, eda, and axin2. Moreover, in our overexpression assays of wild‐type human or zebrafish Wnt10a, we found loss of anterior neural identity in a dose‐dependent manner similar to that reported for overexpression of other canonical Wnt genes (Lekven et al. wnt10a is acting outside of its normal signaling context and that mutations of these conserved residues affect specific regulation of wnt10a signaling in tooth formation, perhaps reflecting regulation of diffusion or coreceptor interaction. As the development of zebrafish teeth is relatively late, MO injections are unlikely to eliminate wnt10a expression completely, and some gene activity is still expected.As zebrafish teeth are continuously replaced throughout life, we focused our analyses to reflect development of the first tooth, usually by 4–5 dpf, and our findings indicate that WNT10A is critical for tooth development and mutations in this gene lead to arrest of normal tooth development in humans and in zebrafish. Moreover, we demonstrate that perturbation in wnt10a expression leads to altered expression of additional tooth development genes suggesting that wnt10a may have direct and indirect roles in tooth development.In conclusion, our findings provide evidence that The authors have no conflicts of interest to declare.Figure S1. WNT10A amino acid alignment across vertebrate species.Figure S2. Analysis of zebrafish wnt10a morphant embryos.Figure S3.wnt10a knockdown causes cartilage abnormalities in zebrafish.Table S1.WNT10A custom‐SNPs used for genotyping.Table S2. Morpholino and primer sequences used in this study.Table S3. Number of embryos with and without teeth.Table S4. PCR primers used for wnt10a overexpression and analysis of mutant alleles.Click here for additional data file.


Following cerebral arteriovenous malformation (AVM) surgery, severe brain edema and hemorrhage may be caused by postoperative normal perfusion pressure breakthrough (NPPB). Sedation is necessary for this population. It is a challenge for the anesthesiologist to maintain hemodynamic stability without interfering with the neurological assessment. In Japan, propofol is contraindicated for pregnant patients. Dexmedetomidine is a versatile drug in anesthesia practice and may be useful for this situation. There is no report using dexmedetomidine for the purpose of NPPB control in pregnant patients. We describe the postoperative management with dexmedetomidine for a pregnant patient who underwent cerebral AVM nidus removal.2 in air and sevoflurane. The AVM was completely removed, and no perioperative complications occurred. Postoperative sedation with dexmedetomidine was used to prevent breakthrough hyperperfusion and cerebral edema.A 32-year-old patient presented with headache at the 16th week of gestation. Neuroimaging revealed an intraventricular hemorrhage and an AVM at the right anterior horn of the lateral ventricle which caused bleeding. A multidisciplinary team discussion was done, and then a craniotomy for AVM nidus removal was performed under general anesthesia. Preanesthetic aspiration prophylaxis and rapid sequence induction were added to our conventional anesthetic management. Hypotension occurred after anesthetic induction but the patient recovered by volume resuscitation and vasopressors. Anesthesia was maintained with 50% ODexmedetomidine infusion was used for postoperative sedation without causing any side effects, and it can be an alternative for sedation, especially when propofol is contraindicated. Neurosurgical intervention during pregnancy is still a challenge due to the strict surgical indications and maternal-fetal effects of anesthetic drugs. Arteriovenous malformation (AVM) accounts for about 50% of subarachnoid hemorrhage in pregnant women . It has A 32-year-old secundigravida presented with constant headache and complained of disorientation in her day-to-day life at 16 weeks of gestation. Because her headache was not controlled with 400 mg of acetaminophen, she visited a nearby hospital at week 17 of gestation. She was conscious and alert. She was not hypertensive; her heart rate (HR) was 83 beats/min and her blood pressure (BP) was 103/50 mmHg. Obstetric ultrasonography showed a single viable fetus without any fetal compromise. Computed tomography (CT) and magnetic resonance imaging revealed intraventricular hemorrhage, but there was no evidence of increased intracranial pressure. Three-dimensional contrast-enhanced computed tomography angiography showed a 1.5 cm AVM nidus fed from a branch of the right Heubner’s artery and intraventricular venous drainage. At week 18 of gestation, she was referred to our institution for further treatment. Her AVM was classified as grade 2 in the Spetzler-Martin grading system. After a preoperative multidisciplinary conference including neurosurgeons, obstetricians, and anesthesiologists, AVM nidus removal was planned at week 21 of gestational age under general anesthesia.2 of 33–40 mmHg, and BIS value of 40–60. The AVM was completely and successfully removed. Estimated blood loss was 50 mL, and 850 mL of bicarbonate Ringer’s solution and 1000 mL of hydroxyethyl starch were infused. The total duration of the surgery and anesthesia was 186 and 259 min, respectively. FHR monitoring confirmed fetal viability at the end of anesthesia. In consequence, dexmedetomidine 1 mcg/kg was administered over 10 min as a loading dose then followed by a maintenance infusion of 0.7 mcg/kg/h. After the dexmedetomidine infusion was initiated, her vital signs were stable without hypotension or bradycardia. Complete resection of the AVM without intraventricular hemorrhage and cerebral ischemia were confirmed by postoperative CT. In the intensive care unit (ICU), she was awake while the maintenance infusion rate of dexmedetomidine was 0.35 mcg/kg/h. One hour after surgery, the patient recovered fully awake without neurological deficit. Dexmedetomidine infusion was then stopped, and extubation was performed. The total dexmedetomidine dose used was 99.5 mcg, and the duration of infusion was 2 h. Multimodal analgesia of intravenous fentanyl and acetaminophen was effective to control postcraniotomy headache without postoperative nausea and vomiting. Her analgesic requirement was reduced, and her postoperative pain score was 0. She was discharged from the ICU on postoperative day 1 and went home on postoperative day 10. There were no obstetric complications, and the pregnancy proceeded uneventfully.The patient was premedicated with famotidine 20 mg intravenously. Standard cardiorespiratory monitoring, invasive BP, and bispectral index (BIS) monitoring were adopted. Left uterine displacement was done, and the fetal heart rate (FHR) was reassured before the induction of anesthesia. After preoxygenation, rapid sequence induction with cricoid pressure was performed with fentanyl 150 mcg, thiopental 250 mg, and rocuronium 50 mg. Intubation was successfully done on the first attempt with a McGRATH™ MAC video laryngoscope. Maternal BP decreased to 83 mmHg after the anesthetic induction, but it subsequently recovered with volume resuscitation and vasopressors that included ephedrine 4 mg and two boluses of phenylephrine 0.05 mg. Anesthesia was maintained with 50% oxygen in air, sevoflurane, and remifentanil 0.3–0.5 mcg/kg/min. End-tidal concentration of sevoflurane was titrated according to the depth of anesthesia. During the operation, the systolic BP was maintained in the range of 90–120 mmHg, HR of 82–86 beats/min, peripheral oxygen saturation of 99–100%, an end-tidal COAfter an excision of a high-flow AVM, there is the potential risk for postoperative neurological complications due to NPPB which leads to hyperemia, cerebral edema, and intracranial hemorrhage without evidence of residual AVM . PatientIn postoperative sedation of obstetric patients, no sedative drug has been reported as the drug of choice due to a lack of adequate or well-controlled studies. Propofol may be associated with lower initial Apgar scores at the time of cesarean section and may cause mild metabolic acidosis during a long neurosurgical procedure in pregnant patients. Moreover, in several countries, including Japan, propofol is stated by the manufacturer to be contraindicated during pregnancy , 6. BenzDexmedetomidine is a highly selective alpha-2 adrenergic agonist which is a useful agent because it provides anxiolytic and sympatholytic effects, analgesia, and conscious sedation without respiratory depression. The existing literature reports on dexmedetomidine use in pregnant patients who underwent neurosurgery are limited. Recently, Handlogten and colleagues revealed that dexmedetomidine infusion allowed adequate sedation for an awake neurosurgical procedure in a pregnant patient without causing any adverse effects . For preDexmedetomidine is considered a multipurpose agent for pregnant patients who undergo neurosurgery. It is a sedative agent with minimal effects on respiratory function, maintains hemodynamic stability and cerebral protection with analgesic effects, and also decreases the risk of postoperative complications. Because propofol is prohibited for maternal use in Japan, our case showed that dexmedetomidine is an alternative to propofol for sedation in the obstetric population.


Spontaneous rupture of a continent cutaneous urinary diversion is uncommon and diagnosis of this situation requires a high degree of suspicion. In this paper we present a 66-year-old man with continent cutaneous pouch after radical cystoprostatectomy that presented with spontaneous perforation 25 years after surgery. Spontaneous pouch perforation in our case after 25 years emphasizes the need for long follow-up in patients with continent diversion. Spontaneous rupture of a continent cutaneous urinary diversion is uncommon and diagnosis of this situation requires a high degree of suspicion and emphasizes the need for long follow-up in patients with continent diversion.The 7th most common malignancy in males and 17th in females is bladder cancer . The besWhile orthotopic bladder replacement is accepted as a diversion method, but continent cutaneous pouch for reconstruction of the lower urinary tract is a good option in suitable cases after radical cystectomy  and the Some complications which are specifically associated to catheterizable cutaneous pouch consist of catheterizing difficulties, stenosis of stoma, stones, stricture of ureterointestinal anastomosis, and ultimately pouch rupture. Majority of complications take place in the first three years after surgery however, rarely some events may occur many years after the operation ,10.Spontaneous perforation of continent cutaneous urinary diversion is an absolute rare event; yet it is a well-documented complication after reconstruction of lower urinary tract .A 66-year-old man presented to the emergency ward complaining of fatigue, malaise and abdominal pain without signs of generalized peritonitis from two days ago. The patient was ill and conscious – but not toxic – without any fever or changes in blood pressure and heart rate. The patient experienced unwillingness drinking and eating and simultaneously decrease in urine output. Laboratory data showed increasing in blood urea nitrogen (BUN) and creatinine (Cr) with mild metabolic acidosis. Serum Cr: 1.9 mg/dL, BUN: 71 mg/dL, ph: 7.24, PHCO3: 11 mm Hg and Pco2 was 33 mm Hg. Ultrasonography of abdomen and pelvis revealed no hydronephrosis, however some intraabdominal free fluid was detected.He had a continent cutaneous urinary diversion with detubularized ileum due to radical cystoprostatectomy 25 years ago after diagnosis of muscle invasive bladder cancer.After the surgery, clean intermittent catheterization (CIC) has been conducted every 6 hours to make empty his neobladder. During the first year post-surgery, he has under follow up surveillance and there were no metastasis and tumor recurrence during this period, however he abandoned follow up and had experienced no problem for 25 years until 2 days ago.At entrance immediately hydration and antibiotic therapy started and a 16 Fr Foley catheter gently inserted in the pouch through the cutaneous stoma and ultimately only 500 mL of urine was drained. Examination revealed periumbilical pain and tenderness without rebound tenderness. Patient mentioned no defecation or gas passage during last 2 days. A computed tomography (CT) with oral contrast revealed no bowel obstruction, while it also indicated intraabdominal free fluid as like ultra-sonographic findings . The CT-While on conservative managements, his abdominal distention and decreasing in urinary output was progressed, we decided to explore the abdomen. At laparotomy a 2 cm defect in left anterior side of the pouch was detected and repaired.His postoperative course was passed without any complication and he discharged one week after surgery while, Foley catheter remained inside the pouch via the cutaneous stoma. After three weeks Foley catheter was removed and he continued CIC.Complication rate is high in urinary diversion even after 5 years; Scott et al concluded that the patients should be informed of complications and they should be under followed up as tightly as for cancer follow up . AccordiSpontaneous rupture of continent urinary diversion is extremely rare late complication though in some studies this complication is not even reported at all . In factThe rate of complications should be chronically evaluated while the proportion of patients decrease during the following years, hence it might be underestimated years or decades after surgery .Mansson et al detected a higher rate of neobladder perforation in continent cutaneous urinary diversion than orthotopic pouches, while there is no pop-off mechanism in the former. The incidence of perforation in this report is 1.5% and there is no difference between colonic or ileal pouches in perforation rate. Although there was a perforation in appendiceal outlet in one case .Most complications of radical cystectomy and urinary diversion is related to the urinary diversion section. Although the literature reports all complications are the same for the two procedures .Some crucial contributing factors for rupture are possible weakening and ischemic changes of pouch wall due to acute or chronic over-distention of continent diversion .Patients may present with some symptoms such as localized pain around stoma or mild to severe generalized abdominal pain or signs of frank peritonitis, sepsis, ileus and fever. Admittedly diagnosis needs a high degree of suspicion. Computed tomography with pouchography is possibly a better option for imaging.Furthermore, several reports of conservative management with catheter drainage, and wide spectrum antibiotic therapy in literature were reported , howeverSpontaneous pouch perforation in our case after 25 years emphasizes the need for long follow-up in patients with continent diversion. Sudden onset of abdominal pain in a patient with continent pouch (orthotopic or cutaneous) should be considered rupture until proven otherwise. To our knowledge, this is the latest spontaneous pouch perforation that reported till now.Authors would like to thank Dr. Zohre Kiani for his invaluable comments on the manuscript.SYH; conceived the study. MD; participated in design and coordination of the study. AFM; collected data and wrote first draft. ZS; helped in data collection and drafting the manuscript from beginning to the last point. MA; read and approved the final version of manuscript. All authors also read and approved the final manuscript.The authors declare no conflict of interest.Ethical issues  have been completely observed by the authors. Written consent was obtained from the patient for publication of the study.None.


Cultural orientation is defined as an individual’s cultural preferences when encountering imported culture while still living in the native culture. Data was analyzed from 1305 Chinese university students attending universities in Beijing, Kunming, and Wuhan. Cultural orientation was assessed with the Chinese Cultural Orientation Questionnaire, which assesses both Western and Traditional Chinese cultural orientations. The analysis used hierarchical logistic regression with nondrinkers as the reference group and controlling for demographic factors . Western cultural orientation was found to significantly increase the odds of recent drinking. The results indicated that higher Western cultural orientation was, after gender, the second most important factor associated with Chinese college student drinking frequency. Traditional Chinese cultural orientation was not associated with drinking frequency. This study highlights an unexpected outcome of globalization on students who have not left their home cultures. Traditionally in China, drinking alcohol is both a normal part of the daily diet, especially in rural areas, and an important part of rituals, business occasions, festivals, and special events. Moderate drinking on important occasions is encouraged, but excessive drinking is discouraged through various social sanctions. The ability to consume alcohol without visible effects is admired, but visible intoxication is considered a personal shortcoming and a disgrace for one’s family and colleagues –3. The oCultural orientation is an individual’s identification with traditional cultural values and imported foreign cultural values. Cultural orientation reflects an individual’s cultural preferences as he or she encounters influences imported from an outside culture, such as Western values, while still living in and surrounded by his or her native culture. Foreign influences typically come from mainstream and social media, travel, contact with foreigners, use of foreign products, and trying foreign foods, fashions, and celebrations. As a result, a person’s core cultural values (orientation) are challenged and may change. Cultural orientation differs from acculturation, which occurs when an individual emigrates to another country and adopts aspects of the new country’s culture. Today, globalization makes changes in cultural orientation more likely, especially in countries like China that are experiencing new and increasing exposure to foreign influences. Some researchers  suggest Cultural orientation and its contribution in explaining some of the variances in individual alcohol use by young people was first reported the 1990s by Eide and Acuda –9. Eide beyond prominent known influences, we controlled for age [The current study examined the associations of cultural orientation with alcohol use among Chinese university students. The principal hypothesis for this study was that a more Western cultural orientation would be associated with more frequent drinking. Our objective was not to propose cultural orientation as the most important factor in explaining drinking patterns, but to provide a different perspective on the role of cultural influences in drinking behavior. Many variables influence drinking behaviors. To examine the association of cultural orientation with drinking  for age , gender[ for age  and rura for age .The results of this study are both empirically and practically significant in an era of increasing globalization. Empirically, they expand our understanding of the associations between cultural influences and drinking behavior among university-aged students. Previous studies of cultural orientation in China have primarily focused on high school students–12, and The ethical review for research with human subjects was conducted by the IRB of the University of Nebraska-Lincoln . The UNLIRB waived informed consent procedures. Officials from the participating universities in China approved the study.A total of 1,439 students completed the questionnaire. Students were enrolled in 13 universities in Beijing (northern China), Kunming (southwestern China), and Wuhan  and were from hometowns all over China. Trained data collectors gathered the data in existing undergraduate classes. All students in attendance on the survey day completed the survey. The survey, written in Chinese, included a cover page informing the students of the purpose of the study, instructions on how to complete the scales, an assurance of anonymity and confidentiality, a statement of their right to not answer questions without affecting their standing, and a notification that the university authorities had approved the project. Approximately equal numbers of students in each university completed the survey.Prior to analysis, questionnaires with missing responses on more than half of the cultural orientation items or with contradictory answers in the alcohol use questions were eliminated. The total remaining questionnaires 1,305 (91%) were included in the analysis. There was no specific pattern of demographics in the 134 surveys dropped from the analysis that would affect the result of the analysis. The final sample was 40% male and 60% female, ages between 17 and 30 years . Of these, 62% were freshmen, 15% sophomores, 11% juniors, 6% seniors, and 6% graduate students. Sixty-four percent of the students were from urban hometowns (cities and counties), and 35% were from rural areas (towns and villages).The instrument used in this study was a revised version of the Chinese Cultural Orientation Questionnaire (CCOQ) developed by Shell et al. and Xue respect for elders , filial piety (“I would do whatever my parent wants me to do”), pride in being Chinese (“I am proud of China’s long history”), collectivism , and harmony (“I won’t show off my strengths to avoid upsetting others”). The Western cultural orientation factor was measured with four sub-factors: dating attitudes (“I feel uncomfortable seeing couples show intimacy in public”), interest in Western culture (“I appreciate the context of Western culture that emphasizes freedom and taking it easy in life”), consumerism (“I would rather buy something that makes me happy than deposit money in the bank”), and personal appearance preference (“I like to dye my hair like Westerners’ hair”). A confirmatory factor analysis with these nine first-order factors and two second-order factors was used. Based on Hu & Bentler’s[p< .05 and CFI = .673 did not show good fit; however, χ2 is sensitive to sample size and model complexity. The SRMR with one other fit index was adequate for model evaluation based on the two-index presentation strategy). In addition, the correlation between the two cultural orientations was nonsignificant, confirming the bidimensional model theory that assumes an individual’s orientations toward the two cultures are independent. Therefore, the two second-order cultural orientations were used as two separate continuous variables in the following analysis . FactorDrinking frequency was obtained by asking students to report their drinking in both the last 30 days and the last 12 months. Students were divided into three drinking categories: nondrinkers , drinkers (had drunk in the last year but not in the last 30 days), and recent drinkers . This classification is broad, resulting in only three drinking categories, but our experience in China has been that narrower definitions become increasingly inaccurate for a number of reasons. In China, there is no standardized alcohol serving size, no standardized alcohol container size, greater variability in the percentage of alcohol by volume in different drinks, and drinking practices involving frequent topping up, mixing different drinks on a drinking occasion, and encouraging others to drink, making it difficult to recall what was actually consumed ,22. DrinDemographic questions asked students for their gender, age, and hometown .F = 8.22 for male; Mean = -.01, F = 5.93 for urban) and higher Western cultural orientation .A one-way ANOVA  showed tM = .07, F = 20.07), but no significant difference in their Chinese cultural orientation.For the drinking behaviors, 19% of the sample had not consumed alcohol in the 12 months (nondrinkers), 42% had consumed alcohol in the last 12 months but not in the last 30 days (drinkers), and 39% had consumed alcohol in the last 30 days (recent drinkers). The three types of drinkers showed significant differences in Western cultural orientation, with the recent drinkers group having the most Western orientation , gender/being male , and urban hometown  all significantly increased the odds of recent drinking. In step 2, after the two cultural orientation variables were added, age  and gender/being male  were still significant predictors of drinking behaviors. Therefore, compared with the odds of nondrinking, participants’ odds of recent drinking increased by 1.14 times with each increase in age of one year; the odds of male students being recent drinkers were 3.80 times those of female students. Western cultural orientation  also significantly increased the odds of recent drinking, by 3.71 times for every point scored higher on the Western cultural orientation scale. By contrast, Chinese cultural orientation did not significantly associate with the odds of recent drinking . Hometown became non-significant  after adding the two cultural orientation variables in step 2.When comparing a student’s odds of being a drinker with the odds of being a nondrinker, none of these variables was significant in either step 1 or step 2. However, as seen in step 1, when comparing the odds of recent drinking to nondrinking, age Click here for additional data file.


In esotropia with larger angles > near than at distance, splitting of the medial rectus muscle has been suggested as a treatment option. Previous reports of bilateral medial rectus Y-splitting as a first intervention showed a reduction of the distance/near disparity with fewer side effects compared to posterior fixation surgery. We address whether a medial rectus Y-splitting as a secondary and/or a unilateral procedure also reduce distance/near disparity.  We retrospectively reviewed the charts of four patients undergoing Y-split recession as a second and/or unilateral surgery. Main outcomes were distance/near disparity and squint angles.  Three of the four patients had undergone unilateral Y-splitting of the medial rectus as a secondary surgery, three as a unilateral procedure. Mean distance/near disparity was reduced from 17 PD preoperatively to zero at the final follow-up (FU). Preoperative angles ranged from 45 PD to 66 PD at near and from 25 PD to 55 PD at distance. At the final FU, these angles ranged from 0 PD to 20 PD at near and at distance. Mean FU was 42 months (range: 12–60 months).  Y-split recession as a secondary and/or unilateral surgery for distance/near esotropia can reduce distance/near disparity with good long-term results. Residual esotropia can be corrected by adding resection of the lateral rectus muscle. In 1991, Bagolini et al. suggested  in patieTo our knowledge, there has been no previous report on Y-splitting of the medial rectus muscle as a reoperation and/or as a unilateral surgery.An approval of the Institutional Review Board to undertake this study was obtained in December 2012. The study was conducted in adherence to the Declaration of Helsinki.We retrospectively reviewed the medical charts of patients who had undergone strabismus surgery between 2008 and 2012. Patients who received Y-split recession of the medial rectus muscle as a secondary or as a unilateral procedure at our institution were identified.The main outcome measures were the reduction of the distance/near disparity, the squint angle at near and at distance, and adduction and abduction measured with the Kestenbaum Limbus test, whichever is available. We included data collected preoperatively and at postoperative weeks one, twelve, and at the final follow-up (FU).The surgical technique is described elsewhere by Hoerantner et al.  and is sn = 3) or a unilateral procedure (n = 3). At the time of surgery, all patients had worn their full cycloplegic correction for at least 3 months.4 patients were identified as having undergone a Y-split recession of the medial rectus muscle as a secondary procedure . The patient could not remember her early strabismus history. At the age of 6 years, she had undergone strabismus surgery on the left eye (no details known), and four years later, the medial rectus muscle of the right eye had been treated with a posterior fixation surgery according to Cüppers. The squint angle, measured with the alternate prism cover test, was 60 PD at near and 45–50 PD at distance. She also had a hypotropia of the right eye of 20 PD at near and at distance. Bagolini was negative at near and at distance. In the following visits, varying angles of 55–66 PD were measured at near, and of 45–55 PD at distance. At the last preoperative visit, the angle at near was 66 PD and 50–55 PD at distance . SnellenThe forced duction test, performed intraoperatively, revealed a restriction in the abduction of the right eye, which was less severe in the left eye. A unilateral surgery was performed: The right medial rectus muscle was revised and recessed with the Y-splitting procedure, and a 5 mm resection of the right lateral rectus muscle was performed. 1 week later, the angle had decreased to a manifest 30 PD at near and 1 PD at distance. At the last FU at 36 months, the distance/near disparity was resolved. On right fixation, she had an esotropia of 16 PD at near and at distance with a left hypertropia of 25 PD due to the DVD. On left fixation, the angle at near and at distance was 10 PD with a left hypertropia of only 4 PD. Left fixation was assured by fitting the glasses with an additional +2 dpt spherical correction on the right side. At the last FU, the Bagolini test was positive at distance and at near with the right eye performing a corrective outward saccade. The patient was satisfied with the outcome and thus no further treatment was planned.Patient 2 underwent a Y-split recession at the age of 15 years.Her congenital esotropia had been treated elsewhere at the age of 2 years with a bilateral medial rectus recession of 7 mm, combined with a bilateral inferior oblique recession. When she first presented to our hospital at the age of 3, years an alternating esotropia and a DVD remained. Under full cycloplegic correction, varying angles up to 12 degrees were measured. As the cosmetic situation was considered satisfactory at conversation distance of 2 meters, emphasis was placed on visual development. She was followed closely and a slight amblyopia of the left eye was successfully treated with occlusion therapy.At the age of 15 years, the patient opted for further strabismus surgery. Visual acuity at distance and at near was 1.25 OU. Testing of binocularity with the Bagolini test showed suppression of the left eye. At that time, she was under full cycloplegic correction with a squint angle of 50-51 PD at near and 27–29 PD at distance. The angle at near could be reduced to the distance angle by a near addition of +3 dpt. This was, however, not tolerated by the patient. A unilateral Y-split recession of the medial rectus muscle of the left eye was combined with a 5 mm resection of the left lateral rectus muscle. At the last FU at 60 months, distance/near disparity was resolved and only an esophoria of 6 PD remained at distance and at near fixation as well as a small DVD that was well compensated. Surprisingly, binocular functions developed: the Bagolini test was positive at distance and at near with the right eye performing a small corrective saccade from superior. Motility was unimpaired. The patient was satisfied with this long-term result.Patient 3 underwent a Y-splitting procedure at the age of 18 years. He had first presented at the age of 18 months with congenital esotropia of about 18 degrees and an amblyopia of the left eye. After sciascopy with atropine, full cycloplegic correction was given, with insignificant reduction of the squint angle. Amblyopia treatment was initiated. Due to poor compliance, the treatment was unsuccessful and subsequently discontinued, as the child developed excentric fixation. Regular examinations followed and at the age of 18 years, the patient inquired about surgical correction of the squint angle, with surgery to be performed only on his left amblyopic eye. Visual acuity for the right eye was 1.25 at distance and at near, for the left eye 0.2 at distance and 0.063 at near. The angle was 42–45 PD at near and 20–25 PD at distance . A unilaPatient 4 underwent a bilateral Y-splitting procedure at the age of 10 years.She suffered from an infantile alternating esotropia with nystagmus, and V-symptom and strabismus sursoadductorius were seen on both eyes. At the age of 4, this had been treated surgically with a 5 mm medial rectus recession, and a 7 mm lateral rectus resection in the right eye and a bilateral inferior oblique recession. 5 years later, wearing full cycloplegic refraction, she presented with a persisting squint angle of 45 PD at near and 30 PD at distance as well as a sursoadduction of the left eye. Snellen acuity was 1.0 at distance and at near OU. She underwent bilateral Y-split recession of the medial rectus muscles in combination with a revision of the inferior oblique muscle of the left eye. 1 week after surgery, the angle decreased to 38 PD at near and 25 PD at distance . At the Splitting procedures have a long history in strabismus surgery. Splitting of the lateral rectus muscle in Duane syndrome has been suggested by Jampolsky as early as 1980 –13. Y-spIn the 1990s, splitting of the medial rectus muscle was introduced as a treatment option in patients with a larger strabismus angle at near than at distance (convergence excess) . Since tCompared to posterior fixation surgery, Y-splitting surgery results in a larger reduction of the maximal angle at near and in a narrower distribution of the final angle at distance and at near around “0” . HoerantAnother recently propagated surgical techniques to correct distance/near disparity are recessions of the medial rectus muscles where the new insertion is created in a slanting manner –18. ThesOur small retrospective case series shows that a Y-splitting procedure of the medial rectus resolves the disparity between large angles at near and small angles at distance even when it is done as a secondary and/or as a unilateral procedure. To our knowledge, this has not been reported before.The reduction of the distance/near disparity may take longer than 3 months but was seen in all patients at final FU >1 yr. This is in agreement with reports of medial rectus-augmented recessions, slanted recessions, or recessions with posterior fixation sutures, where long-term results show an increased effect even after one year .Thus, all our patients had a reduction of their esotropia with a larger effect on the angle at near (average reduction: 38.25 PD) than at distance .Y-splitting procedures offer an alternative method to address esotropia with larger angles at near than at distance. It reliably reduces distance/near disparity also in cases of reoperation and in unilateral surgery. Its effects may take a little longer to stabilize than with traditional recession or resection surgery, but its long term effects are stable. Residual esotropia may be addressed by lateral rectus resection.


It was found that the dielectric constant εm, remnant polarization rP and piezoelectric coefficient d33 reach their maximum values near the rhombohedral–pseudocubic phase boundary. In particular, the 0.7BF-0.3BT composition shows large polarization (rP > 20 μC/cm2) and a temperature-stable piezoelectric property . Near the tetragonal–pseudocubic phase boundary, εm and rP decrease, and the piezoelectric property vanishes when the BF content reaches 4 mol %.Solid solutions of (1- However, synthesis of a single-phase BM perovskite ceramic material by conventional synthesized methods is challenging; this may be due to the small radius of the Bi cation  ~0.14 Å . For BF,~0.676 Å . This ma2+ ions with a large radius expands the unit cell, as shown by the compositional dependence of the lattice parameters O3 system, in that the radii of the Sn4+ ions are much larger than those of the Ti4+ ions [c direction for the Ti4+ ions is reduced, as also shown by neutron diffraction data [3+ and Pb2+ cations on the A-site, the behavior is different. The length of the c axis and the tetragonal distortion for BiScO3-PbTiO3 and Bi(Ti1/2Mg1/2)O3-PbTiO3 systems near the PT side decrease slowly with the decreasing PT content [0.5Zn0.5)O3 leads to the increase in the c axis and the tetragonal distortion near the PT region [In addition, the effects of the structure parameters near the BF and BT sides are also different. Near the BF side, the addition of Barameters . The disi4+ ions . Althougion data . The simion data ,10,11. T content ,7. In coT region ,47. A fid = ~420 °C). The increase of BF content leads to the decrease of the electrical parameters near the BT side. The long-ranged ferroelectric order is disrupted when the BF content is only 4 mol %. An understanding of the electrical behaviors in the BF-BT system may help in the search for high-performance Pb-free piezoelectric ceramics based on similar phase boundaries.In conclusion, the polar order evolutions near the R-PC and T-PC phase boundaries in the BF-BT system are different. Near the R-PC phase boundary, 0.7BF-0.3BT exhibits a large polarization and a temperature-stable piezoelectric property (T


Simulation-based training has become an accepted clinical training andragogy in high-resource settings with its use increasing in low-resource settings. Video recordings of simulated scenarios are commonly used by facilitators. Beyond using the videos during debrief sessions, researchers can also analyze the simulation videos to quantify technical and nontechnical skills during simulated scenarios over time. Little is known about the feasibility and use of large-scale systems to video record and analyze simulation and debriefing data for monitoring and evaluation in low-resource settings.This manuscript describes the process of designing and implementing a large-scale video monitoring system. Mentees and Mentors were consented and all simulations and debriefs conducted at 320 Primary Health Centers (PHCs) were video recorded. The system design, number of video recordings, and inter-rater reliability of the coded videos were assessed.The final dataset included a total of 11,278 videos. Overall, a total of 2,124 simulation videos were coded and 183 (12%) were blindly double-coded. For the double-coded sample, the average inter-rater reliability (IRR) scores were 80% for nontechnical skills, and 94% for clinical technical skills. Among 4,450 long debrief videos received, 216 were selected for coding and all were double-coded. Data quality of simulation videos was found to be very good in terms of recorded instances of “unable to see” and “unable to hear” in Phases 1 and 2.This study demonstrates that video monitoring systems can be effectively implemented at scale in resource limited settings. Further, video monitoring systems can play several vital roles within program implementation, including monitoring and evaluation, provision of actionable feedback to program implementers, and assurance of program fidelity. •We collected 11,657 simulation and debrief videos at 320 primary health centers in Bihar, India.•Video monitoring can support monitoring/evaluation and actionable feedback and assure fidelity.•Video monitoring systems can be effectively implemented at scale in resource-limited settings. Simulation effectively improves technical and nontechnical skills of health care providers in a variety of disciplines . As innoVideo recordings of simulated scenarios are commonly used by facilitators during debriefing sessions to provide factual documentation of the simulation, guide conversation, and stimulate reflection and self-guided learning . Video pLittle is known, however, about the feasibility and use of large-scale systems to video record and analyze simulation and debriefing data for monitoring and evaluation in low-resource settings. Ongoing collection of video data to monitor and evaluate maternal and child health simulation-based training programs is needed to ensure program fidelity, show efficacy, and target limited resources. Low-resource settings present unique challenges when using video-based monitoring and evaluation systems at scale because of poor Internet connectivity, remote locations, and infrastructure issues. Direct observation of deliveries, considered the gold standard for showing changes in clinical practice from training, is expensive and often does not capture data on clinical performance during emergency scenarios because of their relative rarity .To provide facilitators the ability to conduct video-guided debriefs and measure changes in technical and nontechnical skills of mentees, we developed a large-scale video monitoring system that evaluates both simulations and debriefs in Bihar, India. The aim of the overall study was to examine changes in technical and nontechnical skills in simulated clinical scenarios in a large-scale quality improvement project. This article describes the feasibility and design of the video monitoring system and the coding process.The project was implemented in 320 primary health centers (PHCs) in Bihar, India, from January 2015 to January 2017. Bihar's approximate population of 115 million makes it the third largest state in India , with apIn 2015, CARE India (CARE), in collaboration with the Government of Bihar, launched a program called Apatkalin Matritva evam Navjat Tatparta translating to emergency obstetrical and neonatal readiness. One goal of this comprehensive system strengthening and quality improvement initiative was to improve maternal and neonatal health outcomes in public health facilities through interventions aimed at improving supply procurement, provider skills and behavior, and quality of clinical care through adult learning techniques .www.prontointernational.org), and the University of Utah partnered with CARE India and the Government of Bihar to integrate PRONTO's highly realistic simulation and team training activities into the Apatkalin Matritva evam Navjat Tatparta program through mobile nurse midwife mentoring at 320 PHCs by a cohort of 120 nurse mentors (NMs), brought into Bihar mostly from outside the state. The aim was to improve the skills of auxiliary nurse midwives/general nurse midwives (mentees) working at the PHC level. PRONTO, in partnership with the University of Utah, developed a Bihar-specific simulation and team training curriculum to complement CARE India's curriculum. The NMs were trained as mentors by CARE and PRONTO. PRONTO trained the NMs to run simulations, facilitate video-guided debriefing sessions, and conduct postevent debriefing after live births. The simulation and team training activities provided mentees with opportunities to practice technical competencies for the management of a variety of neonatal and obstetric emergency cases as well as nontechnical competencies for better teamwork and communication.In 2014, the University of California, San Francisco (UCSF), PRONTO International (PRONTO)  infant manikin to simulate NR.Pairs of NMs were assigned four PHCs to conduct on-site mentoring visits. Each month, NM pairs visited their four assigned facilities for one week. They returned for weeklong visits every month for eight months. The program was implemented in four phases at 80 facilities per phase . During In addition, at two points during each phase, midpoint (after week 3 of mentoring) and endpoint, mentees participated in three simulations  where performance was assessed to objectively measure changes over time.NMs video recorded every simulation and debrief completed at the PHCs on video cameras provided by the program. Video footage was renamed using a prespecified nomenclature, uploaded to a computer by the NMs as a mp4 video, transferred to encrypted universal serial bus drives, and sent to headquarters in the state capital, Patna, via courier. Video footage was transferred to a secure server by the data manager. The data manager followed a standard operating procedure of video selection to distribute video files to two Hindi-speaking video analysts (VAs) with four-year undergraduate degrees in nursing for video coding. Building the video analysis system began with selecting key simulation scenarios and agreeing on associated indicators. A team of clinical, simulation, and team training experts agreed on technical and nontechnical indicators. These indicators were incorporated into code windows, the visual coding interface that displays different codes besides the video being analyzed using Studiocode™ video analysis software  . Expert Selected videos for analysis fell into two time point categories: (a) midassessment and postassessment videos, which evaluated changes over time in mentees' use of evidence-based practices (EBPs) and their ability to identify and manage simulated maternal and neonatal birth complications in NSVD, PPH, and NR scenarios and (b) simulation videos taken during weeks 3, 5, and 7 of mentoring, allowing program staff to give iterative feedback about trends over time in use of technical and nontechnical skills. For weekly simulations, NMs were encouraged to conduct at least three simulations: NSVD, PPH, and NR, but could exceed this and run any of the 31 simulated scenarios included in the curriculum.•Events that occurred repeatedly throughout a simulation were coded for frequency of occurrence .•Time-bound events were those in which there was an event that should follow a previous event in a specific amount of time . VAs pressed a button to automatically calculate the time between the two events.To assess mentee performance during simulations, coding occurred in two passes . During Data quality indicators were also recorded on the code window to reflect when the VAs were unable to hear sufficiently or when the camera was blocked and they were unable to see during the video. In addition, a scenario conclusion indicator was included to record the status of the patient at the end of the scenario as stabilized, referred, further consult needed, died, or scenario stopped.1.www.qualtrics.com/), a web-based survey.Debriefing Assessment for Simulation in Healthcare©  : The DAS2.Center for Advanced Pediatric and Perinatal Education  Debriefing Evaluation Tool© : VAs useTo assess the quality of debriefs conducted by NMs and provide feedback, a random subset of videos was selected for analysis. Two tools determined the indicators for the debrief videos:To ensure consistency of coding and maintain a high level of data quality across the VAs, the research team initially piloted the code windows by triple coding 10 videos. Researchers routinely assessed interrater reliability (IRR) across the two VAs. For each phase, a randomly selected sample of 5% to 10% of the simulation videos was blindly double coded by both VAs and sent to UCSF where IRR scores were calculated in Studiocode™ software. IRR scores were calculated for all indicators individually and averaged for first-pass nontechnical codes and second-pass technical codes. All debrief videos were double coded. The www.iihmr.org) in Jaipur, India. Written informed consent was obtained from all participating NMs and mentees.The overall study design, including the video recordings of each simulation and debrief in Bihar, was approved by the Committee on Human Research at UCSF under study approval no. 14-15446 and the institutional review board at the Indian Institute of Health Management Research (Throughout the four phases of the nurse midwife mentoring program, a total of 11,278 simulation and debrief videos were collected, resulting in an estimated 2,828 hours of video data as described in the Overall, data quality of simulation videos was very good regarding recorded instances of unable to see and unable to hear in phases 1 and 2 of mentoring. Twenty percent of videos recorded one or more instances of being unable to see, whereas inability to see the baby was twice as common as inability to see the mother. In 2% of simulations, VAs reported that they were unable to hear. Video analysis showed that 60% of videos resulted in a stabilized patient at the conclusion of the simulation and 25% were reportedly stopped before the end of the simulation. Reasons for stopping a simulation included needing to attend to a live patient, clinical mismanagement of the simulated case offering the opportunity for a teachable moment, or technical issues such as equipment malfunction.The video monitoring system allowed program implementers to use data to guide programmatic decision-making. Because this program rolled out in four phases, data-informed improvements shaped as lessons and were learned on a phase-by-phase basis. For example, after analyzing phase 1 simulation video data, the program implementers recognized NR as an area for knowledge and skill improvement. Feedback was given to enhance the NM's teaching of NR skills by repeating relevant simulations and by providing a timer to sensitize to the importance of urgency. Secondary analysis of these NR videos revealed the difficulty in assessing effective resuscitation, resulting in a set of new instructions about camera placement. A secondary code window was developed for NR experts to better assess the quality of specific NR practices in birth asphyxia simulations.To ensure mentees received the best training possible, a robust feedback mechanism was developed for NMs about their skills in simulation facilitation and debriefing. The data generated from the video monitoring system were used to develop individualized reports for each NM pair. Reports from simulated cases displayed graphs of mentee performance from previous phases during simulations of NSVD, PPH, and NR. A written interpretation accompanied each graph congratulating NMs on areas in which mentees consistently performed well while encouraging NMs to focus teaching in areas where mentees displayed room for improvement. To give debriefing feedback to NM pairs, program implementers used data collected from the modified CAPE and DASH tools. Furthermore, to ensure reports would strengthen individual-specific teaching, program implementers contacted each NM pair either telephonically or in person to discuss the reports and methods in the hope that this would help NMs enhance their facilitation practices.To our knowledge, this is the first report using video analysis for monitoring and evaluating a maternal neonatal care quality improvement project at this scale and in a resource-limited setting. This article documents the experience of developing and implementing a video monitoring system for clinical quality improvement and demonstrates that video analysis is not only feasible but also a programmatically valuable technique for program monitoring and evaluation at scale in a resource-limited setting. Overall, the video monitoring system implemented was a successful mechanism at providing important feedback to improve and adapt the program on an iterative basis. The system provided data to allow program implementers to measure the use of technical and nontechnical indicators occurring during simulation, supply objective information to guide programmatic decision-making, address technical problems in simulations early, and provide comprehensive feedback to NMs to effectively guide their own simulation facilitation practices.This approach of analyzing nontechnical behavioral indicators warrants highlighting. Evidence is building for the importance of nontechnical skills for effective clinical care and avoiding errors . HoweverUsing video recordings for monitoring and evaluation posed challenges. The process of data collection, coding, and transferring large video files was labor intensive. Although programmatic feedback to NMs was iterative, real-time feedback was not possible. Fortunately, NMs served as mentors for two or more phases of implementation, allowing program implementers to use data from past rounds to subsequently develop relevant individualized program reports. In addition, we have not provided data on the cost-benefit or cost-effectiveness of this strategy, which is important for scale-up interventions.These findings have important implications for scale-up of innovative interventions within government systems. Pilot interventions are often difficult to scale-up , hindereThis study demonstrates that video monitoring systems can be effectively implemented at scale in resource-limited settings. Furthermore, video monitoring systems can play several vital roles within program implementation, including monitoring and evaluation, provision of actionable feedback to program implementers, and assurance of program fidelity.


A rank based social norms model predicts that drinkers’ judgements about their drinking will be based on the rank of their breath alcohol level amongst that of others in the immediate environment, rather than their actual breath alcohol level, with lower relative rank associated with greater feelings of safety. This study tested this hypothesis and examined how people judge their levels of drunkenness and the health consequences of their drinking whilst they are intoxicated in social drinking environments.N = 400) also answered four questions asking about their perceptions of their drunkenness and the health consequences of their drinking (plus background measures).Breath alcohol testing of 1,862 people  in drinking environments. A subset , how extreme they regarded their drinking that night , how at risk their long-term health was due to their current level of drinking  and how likely they felt they would experience liver cirrhosis (b 4.8. 95 % CI 0.7 8.8). People were more influenced by more sober others than by more drunk others.Perceptions of drunkenness and the health consequences of drinking were regressed on: (a) breath alcohol level, (b) the rank of the breath alcohol level amongst that of others in the same environment, and (c) covariates. Only rank of breath alcohol level predicted perceptions: How drunk they felt (Whilst intoxicated and in drinking environments, people base judgements regarding their drinking on how their level of intoxication ranks relative to that of others of the same gender around them, not on their actual levels of intoxication. Thus, when in the company of others who are intoxicated, drinkers were found to be more likely to underestimate their own level of drinking, drunkenness and associated risks. The implications of these results, for example that increasing the numbers of sober people in night time environments could improve subjective assessments of drunkenness, are discussed. Excessive alcohol consumption places drinkers’ health at risk both in the long term and during the drinking episode . In the Whilst intoxicated, people might be assumed to judge their levels of drunkenness based solely on how much they have actually drunk. This intuitive “actual intoxication” approach is perhaps a partial motivation for calls to increase the availability of information on the amount of alcohol consumed, for example through mandatory labeling of bottles with alcohol units .We propose in contrast a rank based social norms model, which we test in a sample of intoxicated individuals through modeling the relationship between people’s objective drunkenness  and their ratings of their drunkenness, as well as the relationship between their BrAC and the attitudes they held at that moment about the health consequences of their drinking. Specifically we hypothesize that when drinking alcohol amongst others people’s perception of their own level of drunkenness is influenced by the perception of their level of inebriation relative to others in their immediate vicinity. Our focus is on social drinking and we make no reference to alcohol consumption that occurs in isolation. The “actual intoxication” approach neglects existing social norms research which indicates that - at least whilst individuals are sober - people are highly influenced by their perceptions of how their levels of drinking compare to those of others within their reference groups e.g., –7). Whil. Whil7])First, it is not clear whether drinkers compare their levels of intoxication to the actual levels of intoxication of those in the same drinking environment, or to their incorrect beliefs about the levels of intoxication of others. Generally, social norms research has shown that people have an inaccurate impression of how much others drink - possibly being motivated by a desire to self-enhance through seeing themselves as relative lower drinkers - and that it is this inaccurate impression that affects judgements of the heaviness of actual drinking , 7. HoweSecond, it is also not clear whether: (a) comparisons to others would bias the basic relationship between objective and subjective drunkenness , or (b) the relationship between objective and subjective drunkenness is wholly based on comparisons to others .Third, the cognitive mechanisms through which people compare their level of drinking to that of others are not known. In making specific predictions for this study, we were guided by independent research from psychophysics which focuses on how people judge the magnitude of stimuli (see ). Such rFourth, if people do compare themselves to others, it is not clear whether their judgements of their own intoxication would be equally, more, or less influenced by people who drink more than they do relative to those who drink less. The alcohol and social norms literature suggests that sober people have a tendency to over-estimate how much others drink, this effect being consistent with a self-enhancement bias motivated by a desire to see one’s consumption as relatively lower e.g., , 7). Thi. Thi7]).In this study we examine for the first time how people judge their drunkenness and the health consequences of their drinking whilst they are intoxicated in social drinking environments. The focus on health was motivated by recent calls for more social norms research to focus on perceptions of the health consequences of personal levels of drinking in addition to simply perceptions of the heaviness of drinking , 23. Bast < 0.1). Of these 477 participants, 39 did not provide gender information and 38 did not answer the covariation questions . This left a usable study sample of 400 participants who completed all relevant measures (representing a 60 % response rate from those asked to participate).This research was a component of a larger twenty-four month study , twelve drunkenness  to 10 (completely drunk) scale?”), (2) extreme drinking  to 10 (completely extreme) scale?”), (3) risk to long-term health  to 10 (definitely will) scale?”), and (4) risk of liver cirrhosis  to 10 (definitely will) scale?” . The key question used to test the theory was the first, with the second used to replicate the finding with alternate wording that stressed personal drunkenness as a function of that respondent’s history of alcohol use. The third question assessed the subsidiary question of how people judge the long term health risks associated with their drinking whilst intoxicated, supported by the fourth which aimed to show that the same results can apply to assessment of risk on a specific as well as a general health evaluation.Four questions were administered to assess perceived drunkenness and potential health risks of the current levels of drinking; (1) The study therefore made use of two sets of data. All those from whom BrAC was recorded were used in the ranking process. A subset of these participants also completed the risk judgement questions and it was their responses that were used to test hypotheses on the relationship between rank (relative to the larger pool of respondents) and judgements. Finally, our hypotheses were specific to drinkers in the drinking environment. Therefore respondents who yielded a BrAC of 0 μg/100 ml were not eligible for inclusion in the rank-judgement analyses, although they were included in the larger pool from which rank was determined as they were present in the same environment.All aspects of the research presented here were scrutinised and approved by the Medical and Dental Research Ethics Committee, Cardiff University prior to data collection.N = 400), we next calculated a new variable for each individual representing the rank of an individual’s BrAC within the reference group through the formula;i - 1) was compared with the total number of people within that individual’s reference group (n - 1) to provide a relative rank score (Ri) normalised between 0 and 1. The primary test, conducted separately for each of the four outcome questions, involved simultaneously regressing outcome on both BrAC and rank BrAC, including covariates. These covariates critically included how the person’s intoxication differed from the average intoxication of those around them, in order to rule out this rival explanation of how relative comparisons are made. Only rank BrAC was hypothesised to predict each outcome.We tested whether participants’ judgements were predicted by BrAC rank within a reference group using established statistical procedures developed for this purpose elsewhere , 29. UsiSeveral variables (which may have had a confounding effect) were assessed for use as covariates in a planned sensitivity analysis. Session duration might have an independent effect with those who had been drinking longest over-estimating the effects of alcohol through fatigue. Also included was the time of survey which was reduced to before and after 11 pm, a time that denotes a transition from drinking in regular pubs to nightclubs and so distinguishes between early evening and late night drinkers. Finally, FAST scores, which indicate patterns of risky drinking , 31, wern = 183). On average, participants saw themselves as moderately drunk and moderately at risk . Men  yielded higher alcometer scores than women , and a one-way ANOVA yielded a significant main effect of location on BrAC  = 44.0, p < 0.001). These differences by gender and location indicate that there was sufficient variation between the reference groups for rank BrAC and BrAC to provide meaningfully different measures. There was an association between rank BrAC and BrAC . We therefore used the variance inflation factor (VIF) to assess collinearity between the explanatory variables BrAC and Ri. The VIFi is given by (1 – R2i)-1 where R2i is the R2 from regressing the ith independent variable on all other independent variables. The VIF shows how much the variance of the coefficient estimate is being inflated by multicollinearity. A VIFi >10 indicates harmful collinearity [Descriptive data of participants’ responses on all measures are presented in Table inearity . In thisRegression models were used to test hypotheses (see Table η that explains any variance in the outcome variables (perceived risk and intoxication) best; η > 1 indicates an upward bias and that drinkers are more influenced by more intoxicated drinkers whereas η < 1 indicates a downward bias and that respondents are more influenced by more sober drinkers [η an iterative process was used in which values of η from 0.01 to 5 in 0.01 steps were regressed onto outcomes. The value of η yielding the greatest R2 value was then selected for each outcome. For all judgements respondents were more influenced by those who were more sober: perceived drunkenness η = 0.70, extreme drinking η = 0.14, long-term health η = 0.17 and liver cirrhosis η = 0.21. In other words introducing sober people into a drinking environment would be predicted to have greater impact on judgements, making people feel more at risk and more intoxicated, compared to the effect on decreasing feelings of riskiness one might expect if very intoxicated people were introduced into the environment. It appears that drinkers are more self-aware of their own level of intoxication when in the presence of those who are sober.To test whether people were more influenced by those who ranked above or below them, the regressions in Model 2 were re-run but with the rank amongst the eight location by gender categories formed through a version of Eq. drinkers . To deteThe study sheds new light on how people judge their drunkenness and the health consequences of their drinking whilst actually intoxicated in real world drinking environments. In such situations the relationship between actual intoxication and drinking perceptions was accounted for by how the person’s consumptions ranked amongst others. Thus, using the same cognitive processes as used in other psychophysical judgements, perceptions of one’s intoxication appear to arise from comparison to others. People are also more influenced by those who are more sober than them, relative to those who are more intoxicated.The research builds on previous social norms work by indicating that people’s judgements of their alcohol use depend on their perception of how intoxicated other people are. The study expands on knowledge by: (a) extending the social norms perspective to judgements made whilst intoxicated in drinking environments; (b) showing that in such settings people are influenced by the actual rather than imagined behaviour of others; (c) showing that in this context people are more influenced by those who drink less ; and (c) consistent with one other paper , suggestThe finding that people are specifically sensitive to rank position suggests that a basic evolutionary mechanism may be implicated. Animals, ranging from crayfish to monkeys, are generally very sensitive to rank position within a hierarchy , and in All studies that study alcohol use in context face numerous difficulties, challenges and are therefore limited. This is usually offset by the considerable advantages of studying behaviour in situ. Never-the-less, this paper has several limitations. First, the operationalization of a social network here assumes those in the same environment who are consuming alcohol influence one another. This may not be consistent with the generic social network approach, in as far that many of those in this study probably have no social relationship. However, those surveyed will have been aware of other people’s presence at least visually. Given the marked effect of alcohol on appearance, such as alcohol-related ataxia, visual cues will play an important role in the formation of judgements. This does warrant further investigation and studies might consider the ecology of influences from immediate social groups through to those in the same venue and environment. Furthermore, the measure for rank intoxication could potentially be subject to less measurement error than the more objective BrAC and this is a concern when measures are correlated , as is tWhile this paper is primarily interested in the manner in which context, or the reference set comprised of other drinkers, informs judgements of being at risk from alcohol consumption, there are obvious links with the traditional literature on perceptions of intoxication generally. For example, self-rated intoxication becomes less accurate as the level of intoxication increases . CertainBrAC, breath alcohol content; CI, confidence interval; FAST, fast alcohol screening test; ml, millilitre; Ri, relative rank score; VIF, variance inflation factor; μg, micrograms


Escherichia coli was accomplished. Sixteen different steady‐state two‐ligand experiments were statistically analysed simultaneously so that the same rate equations and same rate and dissociation constants applied to all experiments. The so‐called rapid equilibrium segments procedure was used to derive the rate equations. The final best‐fit mechanism included the normal activation and transfer steps, and reorganization of the steps between them and after the transfer step. In addition, the analysis strongly suggested an additional activation step, formation of a new isoleucyl‐AMP before the isoleucyl‐tRNA was freed from the enzyme. The removal of Ile‐tRNA was possible without the formation of Ile‐AMP if both isoleucine and ATP were bound to the E‐Ile‐tRNA complex, but this route covered only 11% of the total formation of Ile‐tRNA. In addition to the Mg2+ in MgATP or MgPPi, only two tRNA‐bound Mg2+ were required to explain the magnesium dependence in the best‐fit mechanism. The first Mg2+ could be present in all steps before the second activation and was obligatory in the first reorganizing step and transfer step. The second Mg2+ was present only at the transfer step, whereas elsewhere it prevented the reaction, including the activation reactions. Chloride inhibited the IleRS reaction, while 100 mm KCl caused 50% inhibition if the ionic strength was kept constant with K‐acetate. The Kmapp (tRNA) value was increased from 0.057 to 1.37 μm when the KCl concentration was increased from 0 to 200 mm. The total rate equation helps to understand the reaction route and how the simultaneous presence of Ile‐tRNA and Ile‐AMP can cause new possibilities to proofreading mechanisms of this enzyme.Aminoacyl‐tRNA synthetases join correct amino acids to their cognate tRNA at the start of the protein synthesis. Through the kinetic analysis, it is possible to estimate how their functional details correspond to the known structural features. Kinetic analysis of the isoleucyl‐tRNA synthetase (IleRS) from EC 6.1.1.5)Isoleucyl‐tRNA synthetase ( The basic mechanism was elucidated quite early after the discovery of the aminoacyl‐tRNA synthetases Ile (0.9 μm), 3 mm Mg‐(acetate)2 (1 mm excess Mg2+), 50 mm K‐acetate, 1 mm dithiothreitol and the enzyme. The reaction temperature was 30 °C. 180 μm Mg2+ was carried to the reaction mixture by tRNA, and was taken into account in the calculations. The reaction was stopped by pipetting a sample (40 μL) onto Whatman 3MM paper placed close to the surface of 50% formic acid, which reduces the pH below 4 in seconds. The paper pieces were washed three times with a solution containing 0.2 m HCl and 5% acetic acid and finally once with ethanol. The paper pieces were counted for radioactivity of the formed aminoacyl‐tRNA. The initial rates were calculated using the integrated Michaelis equation containing the product inhibition The rate of the aminoacylation of tRNA was assayed with the filter paper–acid precipitation method i exchange activities were measured in a similar reaction mixture to the aminoacylation, but 32PPi  was substituted for the radioactive amino acid and 50 μm nonradioactive PPi was added. The Ile concentration was 50 μm. The product radioactive ATP was separated from the radioactive PPi by paper chromatography as described previously The ATP‐PPThe equations were derived as described previously C and D terms were defined to express the total enzyme intermediate concentrations in the segment (Di*[Ei]) and the rates between the segments (Cij*[Ei]).The total reaction scheme was divided into 4–6 segments, depending on the mechanism to be tested. The enzyme intermediates within a segment should be in equilibrium with each other. The i exchange reaction and the aminoacylation of tRNA using the C and D terms. The segments were handled like the enzyme intermediates in the normal derivation of rate equations.The steady‐state rate equations were derived for the ATP‐PPC and D terms were expressed using the real rate and equilibrium constants and ligand concentrations.In the rate equation algorithm, the The algorithm and the rate equations (from step 2) were used to obtain the calculated rate values. These calculated rate values and the measured rate values were used in the best‐fit analysis.The best‐fit analysis was performed using the nonlinear regression with the least squares of the residuals between the measured rate and calculated rate values. The kinetic constants were refined by successive iterations. The variances and standard errors were calculated as described i exchange or the aminoacylation of tRNA is measured. The experiments were chosen to cover the different parts of the total reaction. For instance, the changes in the ATP and isoleucine concentrations (in segment 1) or PPi concentrations (segment 2) or AMP concentrations (segment 3) describe mainly the events close to the segments where these ligands are bound. In the aminoacylation reactions  and the inhibition dependences as Dixon plots (1/v vs. i). Due to the complexity of the reaction, the Hanes and Dixon plots are seldom straight lines and the v vs. [Mg2+] plots are not hyperbolas.The kinetic measurements for the statistical analysis are presented in Figs  segment  or AMP ci exchange and aminoacylation rates, vexch/vacyl, was determined, and these results were used in the statistical analysis. This relation gives a simple expression if the second activation reaction does not exist In Fig. In the best‐fit analysis, the sum of the squares of the residuals between the measured rate and calculated rate values was used to calculate the error percentages for all two‐ligand experiments in Figs The scheme of the best‐fit model of the reaction is given in Fig. kcat = 0.7 s−1) allowing the equilibrium to be settled in the different segments. The reaction can run either through segment 5 → segment 1 or through segment 5 → segment 6 → segment 2. The rate equation is an algorithm which is shown in the scheme shown in Fig. i exchange in the presence of tRNA and in the absence of tRNA is in Eqn D3 and Ile‐tRNA is not in D4, D5 or D6.The total rate is slow *(1 + [Mg2+]/KMR2))[tRNAf]*([Mg2+]/KMR)[MgtRNA] = [tRNA2tRNA] = [tRNAf]*([Mg2+]/KMR)*([Mg2+]/KMR2)[MgC12 = k3*(1 + [tRNAf]/K5F1 + [MgtRNA]/K51)*[Ile]/K2*[MgATP]/K1MC21 = k‐3*(1 + [tRNAf]/K5F2 + [MgtRNA]/K52)*([MgPPi]/K4M)C23 = k6C*[MgtRNA]/K52*(1 + [MgPPi]/K4M)C32 = k‐6C*(1 + [MgPPi]/K4M)C34 = k6*[Mg2+]/KME3C43 = k‐6*[AMP]/K74*([Mg2+]/KME4)*([Mg2+]/KME42)C45 = k8C*(1 + [Mg2+]/KME4)C54 = k‐8C*(1 + [Mg2+]/KME5)C56 = k35*[Ile]/K25*[MgATP]/KAMC65 = k‐35*[MgPPi]/K4M6C51 = k8*(1 + [MgATP]/KAM+[Ile]/K25) + k8SA*([Ile]/K25)*([MgATP]/KAM) + k8M*[Mg2+]/KME5*(1 + MgATP]/KAM)C62 = k86 + k86P*[MgPPi]/K4M6D1 = ([Mg2tRNA]/K5M1 + (1 + [tRNAf]/K5F1+[MgtRNA]/K51)*(1 + [Ile]/K2))*(1 + [MgATP]/K1M + [ATPf]/K1 + [AMP]/K71)D2 = (1 + [MgPPi]/K4M+[PPi]/K4 + [ATPf]/KA2)*(1 + [tRNAf]/K5F2+[MgtRNA]/K52)D3 = (1 + [Mg2+]/KME3 + [MgPPi]/K4M)D4 = (1 + [AMP]/K74)*(1 + [Mg2+]/KME4)*(1 + [Mg2+]/KME42)D5 = (1 + [Ile]/K25+[Mg2+]/KME5)*(1 + [MgATP]/KAM)D6 = (1 + [MgPPi]/K4M6)PAR5 = C65/C56+C62/C56PAR4 = (C54/C45+C51/C45)*PAR5 + C62/C45PAR3 = C43/C34*PAR4 + C51/C34*PAR5 + C62/C34PAR2 = C32/C23*PAR3 + C51/C23*PAR5 + C62/C23PAR1 = C21/C12*PAR2 + C51/C12*PAR5D1*PAR1 + D2*PAR2 + D3*PAR3 + D4*PAR4 + D5*PAR5 + D6DENOM = D1 to be bound in random order. Mg2tRNA is bound but does not stay bound further in C12 and thus inhibits. Free ATP and AMP compete with MgATP. The binding of the free tRNA (without Mg2+) is weak. The reaction C12 runs both in the presence and in the absence of MgtRNA.Segment 1: The substrates isoleucine, MgATP and MgtRNA are written in i and tRNA can be bound simultaneously, or they do not compete. MgPPi is the reacting form. Mg2PPi is not bound, and its dissociation constant becomes high in the statistical analysis. Mg2tRNA is not bound, either. In D2, the free ATP is written to compete with PPi. It improves the fit, especially in the ATP‐PPi exchange experiments. MgATP does not have the same effect. The binding of ATP to the PPi site exists in the formation of diadenosine tetraphosphate (Ap4A) C23 step, MgtRNA must be present. MgPPi is not necessary, but does not inhibit the step.Segment 2: In the best‐fit system, PP2+ ion is coming to segment 3 in MgtRNA. Another Mg2+ ion is bound to the complex at this segment. The next step, the transfer reaction, requires two bound Mg2+ ions. MgPPi is not necessary in this segment but it does not inhibit either. In the next C34 reaction, MgPPi is not present.Segment 3: One Mg2+ ion must dissociate from the E‐Ile‐tRNA complex in segment 4. The other Mg2+ ion can also dissociate, but can as well be present in the further C45 reaction.Segment 4: AMP and one Mg2+ in segment 5. This can be the same Mg2+ ion as before in C45, but the binding is about 13 times stronger. The Mg2+ ion in segment 5 competes with isoleucine (not necessarily for the same binding site), and the presence of Mg2+ almost totally prevents the further reactions C51 and C56. The further reactions require that both isoleucine and ATP are bound. That is, naturally, obligatory on the route segment 5 → segment 6 → segment 2, but even on the route segment 5 → segment 1 as much as 93% of the reaction goes through the intermediate Ile‐tRNA‐E(Ile)(ATP).Segment 5: The best‐fit mechanism requires strong binding of MgC56 is another activation reaction (like step C12) for the next reaction cycle. The position of the post‐transfer proofreading should be at segment 5, and therefore, D5 in fact is more complicated. Apparently, the synthetic site is opened for new Ile and ATP when the CCA‐Ile end of the tRNA turns to the editing site. The removal of Mg2+(KME5) may have a role in the opening.Step i and Ile‐tRNA from the enzyme at step C62.Segment 6: Both Ile‐tRNA and Ile‐AMP stay attached to the enzyme. The best‐fit analysis suggests a random dissociation order of PPi and AMP.The calculated amounts of the enzyme intermediates in different segments show that 84% of the enzyme is in segment 2 and 6.6% in segment 6 in the absence of PPm concentration of free Mg2+ , which is the concentration of free Mg2+ in the cell Mgfree2+ concentrations (> 10 mm) almost eliminate the chloride inhibition. K‐acetate inhibits weakly but K2SO4 is stronger than KCl .Chloride inhibits the IleRS reaction Fig. . The inhKCl Fig. B. KCl anKm (tRNA) value. The Km values were 0.057, 0.129, 0.334, 0.657 and 1.37 μm at KCl concentrations of 0, 50, 100, 150 and 200 mm, respectively.KCl affects the Km (tRNA) assay, and the experiments similar to Fig. m KCl. 150 mm KCl caused an approximately eight times increase in the value of KMR2 and a twofold increase in KME42. The value of K52 was 0.3, 0.5 or 2.0 μm at 0, 50 and 150 mm KCl, respectively.The best‐fit analysis for chloride effects was performed using three types of experiments: the E. coli. The chloride concentration in growing E. coli is low (below 25 mmThe chloride inhibition in IleRS is somewhat stronger than in the arginyl‐tRNA synthetase 2+ ions in the aminoacyl‐tRNA synthetase reactions 2+ ions in the equations. Only two competition sites were important. Spermidine could replace Mg2+ (KME3) and Mg2+ (KME42), which are involved in the forward and backward transfer reaction. The corresponding dissociation constant values were KSE3 = 470 μm and KSE42 = 390 μm. The competition with Mg2+ (KMR) was much weaker, KSR = 2800 μm. At the other binding sites, no competition was observed.Polyamines can replace part of the Mg2+ (KME5) do not compete, the FOM is 86.6. If [Mg2+]/KME5 is removed from C54, the FOM is 86.8. In that case, Mg2+ (KME5) is not on the same binding site as Mg2+ (KME42), and the backward rate constant k‐8C becomes higher, about as high as k+8C. When Mg2+(KME5) is removed from segment 5, the FOM rises to 108.The above reaction mechanism is the best found so far. If the sum of the error percentages is used as a figure of merit (FOM), it gives the lowest reading, 83.9 per cent units. Some changes can be made in segment 5 without essential weakening the FOM value. If isoleucine and Mg2+ (KME5) is removed, it is 145.When segment 6 is removed, or the reaction runs through segment 5 → segment 1, the FOM is 90, but even then 99% of the reaction runs through the Ile‐tRNA‐E(ATP)(Ile) complex where both ATP and Ile are bound. If ATP is then removed from segment 5, the FOM is 228; if Ile is removed, it is 128; and if MgIn all the above cases, the optimization of all constants was carried out, not only the constants involved in the mentioned change.et al. In one of the oldest kinetic works on IleRS, Yarus and Berg i exchange, but the exchange reaction was continued after cessation of tRNA, in the presence of Arg‐tRNA An analogous case was found with ArgRS, which normally requires the presence of tRNA for the ATP‐PPAlthough IleRS is an one‐subunit enzyme and has one reaction site, it has an additional editing site for the elimination of wrong amino acids C51, or after its formation in C62. The calculated rates show that 89% of the Ile‐tRNA comes through the route segment 5 → segment 6 → segment 2 and only 11% through segment 5 → segment 1 . Moderate changes in the ligand concentrations do not much change this relation. Only PPi lowers the relation; at 50 μm PPi, it is 80%.The present work suggests that Ile‐tRNA is freed from the enzyme either without the formation of Ile‐AMP in reaction i always exist in the aminoacyl‐tRNA synthetase reactions as MgATP and MgPPi, but Mg2PPi seems not to have importance in the IleRS reaction.ATP and PP2+ ions are bound to tRNA 2+ ions are kinetically important 2+ ions are included in the present analysis of IleRS, and one or two Mg2+ ions are attached at the same time to the E‐tRNA complex in different steps of the reaction. The equations contain six Mg2+ ions (in addition to those in MgATP or MgPPi), but some of them must have common binding sites. The terms [Mg2+]/KME3 and [Mg2+]/KME42 must refer to the same binding site as both participate only in the transfer reaction, one forward and the other backward, and in addition, spermidine can replace both of these Mg2+ ions. This binding not only promotes the transfer reaction but also prevents the other steps, including the activation reactions. According to the equation, the ion Mg2+ (KMR2) could have the same site, too, although it represents the binding to tRNA and not to the E‐tRNA complex. Chloride weakens the binding of Mg2+ (KMR2) and Mg2+ (KME42).Generally, several Mg2+ (KMR) and Mg2+ (KME4) form another pair, which probably binds to a common site. They cannot be replaced by spermidine, and chloride does not weaken their binding. In the ordered binding to the free tRNA, Mg2+ (KMR) is bound first, and in the ordered dissociation in segment 4 Mg2+(KME4), it is freed last.Mg2+ (KME4) and Mg2+ (KME5) have the same binding site remains an open question. According to the equations that could be possible, the latter is, however, bound 13 times more strongly. The other possibility of having different sites can also be correct, as removing the term [Mg]/KME5 from C54 leads to only a slightly weaker result in the best‐fit analysis.Whether Mg2+ ion in MgATP or MgPPi, two or three Mg2+ ions participate in the IleRS reaction. One of them can be present in all steps but must be present in the transfer reaction and the reorganizing step before it. The second of them is present only in the transfer reaction and prevents, for example, the activation reaction. A strongly bound Mg2+ ion is involved in the events after the transfer reaction.In conclusion, in addition to the MgIle is eliminated. The post‐transfer proofreading has been shown to occur at a separate editing site where the noncognate amino acids are hydrolysed During the whole time span of studies on IleRS, understanding the discrimination of the noncognate valine has been one of the key objectives In recent years, the pretransfer proofreading has received new attention C65 and C21) or dissociation of the wrong aa‐AMP from the enzyme and hydrolysis thereafter. These rates can be different in the presence of Ile‐tRNA in segment 6 from that in segment 2. In addition, the Mg2+(KME5) ion in the presence of Ile‐tRNA can affect the rates.The kinetic proofreading system has been a subject of discussion and mathematical calculations 2+ ion can have their effects.Still another possibility for the pretransfer proofreading is a hydrolytic elimination of the wrong aa‐AMP on the synthetic site. Even then, the presence of Ile‐tRNA and the MgKm for tRNA is about twice the value of Kp for Ile‐tRNA, or Ile‐tRNA is bound more strongly to the enzyme. The high amount of Ile‐tRNA and its stronger binding mean that the amounts of enzyme intermediates with bound Ile‐tRNA in segments 6 and 5 are higher compared to segment 2. This emphasizes segment 6 as the possible site of the pretransfer proofreading.In the cell, a major part of tRNA is as aa‐tRNA i must be present in C65 and C21. Due to the fast pyrophosphorolysis rates (high k‐3 and k‐35), the required PPi amount is low, estimated to be below 20 μm. High PPi concentrations (0.5 mm) have repeatedly been measured in E. coli cells i concentrations cannot be in contact with the enzyme. PPi increases the pyrophosphorolysis rates in C21 and C65 towards free Ile, and therefore, the advantage of pretransfer proofreading is lost when the corrected Ile from Ile‐AMP is mixed with the original mixture of amino acids.For the kinetic proofreading, some PPThe key aim of the present work has been to do a best‐fit analysis simultaneously on a large number of different steady‐state kinetic experiments and thus improve the model of the reaction mechanism and the accuracy of the equations. Several improvements could be done, although this kind of kinetic work, naturally, cannot address the mechanism at the molecular level but only at the functional level.The procedure of the ‘rapid equilibrium segments’ was used in the work. It can be seen that the equations in the procedure rapidly become complicated when the number of the segments increases. The rate equation algorithm contained 25 separate equations. On the other hand, the aminoacyl‐tRNA synthetases are among the most complicated enzymes with three substrates, three products, activators and editing mechanisms; nonetheless, IleRS could be successfully analysed. The principal advantage of the procedure is that only a limited number of equations in the algorithm must be modified when the details of the mechanism in a given segment are tested.