An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.

Engineering enzyme activity using an expanded amino acid alphabet.

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

A Surgical Technique to Repair Perineal Body Disruption Secondary to Sexual Assault.

Perineal trauma is uncommon in the pediatric population and it is estimated that 5 to 21% is secondary to sexual abuse. We aim to present a proposed surgical technique to repair perineal injuries secondary to sexual assault in female children. The technique is based on the posterior sagittal anorectoplasty (PSARP) for repairing anorectal malformations and, between 2017 and 2019, it was used to treat three girls (2 months, 2 years, and 8 years of age) with fourth-degree perineal injuries secondary to sexual assault. One of them underwent laparotomy and Hartmann's colostomy for an acute abdomen. Two underwent wound debridement and suturing and only had a stoma fashioned at 5 days and 6 weeks posttrauma, respectively. The perineal repair was performed 2, 6, and 7 weeks postinjury and done as follows: with the child prone in jack-knife position, stay-sutures are placed on the common wall between the rectum and the vagina. Using a needle tip diathermy, a transverse incision is performed below the sutures lifting the anterior rectal wall up. Stay sutures are then positioned on the posterior wall of the vaginal mucosa. The incision between the walls is deepened until the rectum and the vagina are completely separated. The deep and superficial perineal body is then reconstructed using absorbable sutures and an anterior anoplasty and an introitoplasty are performed. The stoma in each was closed 6 weeks postreconstruction. At follow-up, now 1 year or more postrepair, all patients have an excellent cosmetic outcome and are fully continent for stools.

Rectal Perforation following High-Pressure Distal Colostogram.

In patients with anorectal malformations and a colostomy, the high-pressure distal colostogram is the technique of choice to determine the type of malformation and thus to plan the surgical repair. Perforations associated with high-pressure distal colostograms are very rare. The aim of our study was to identify pitfalls to prevent perforation secondary to high-pressure distal colostogram. The study included two male patients and was complicated with rectal perforations secondary to high-pressure distal colostogram. Both patients had an imperforate anus without a fistula. One patient had extraperitoneal rectal perforation with progressive contrast spillage into the peritoneum and demised. The other patient developed an extraperitoneal perforation and an associated necrotizing fasciitis of his perineum and scrotum, but he recovered well after debridement. Two further cases of rectal perforation have been described in the literature. Rectal perforation, although rare, is a described life-threatening complication secondary to high-pressure distal colostogram. The cause is excessive contrast pressure. Injection of contrast should be stopped once the distal end of the colon has a convex shape. Intraperitoneal perforation may cause hypovolemic/septic shock, and patients need to be appropriately resuscitated and should undergo laparotomy. Extraperitoneal perforation requires close monitoring for possible local complications, which may necessitate early debridement.

Characterization of imine reductases in reductive amination for the exploration of structure-activity relationships.

Imine reductases (IREDs) have shown great potential as catalysts for the asymmetric synthesis of industrially relevant chiral amines, but a limited understanding of sequence activity relationships makes rational engineering challenging. Here, we describe the characterization of 80 putative and 15 previously described IREDs across 10 different transformations and confirm that reductive amination catalysis is not limited to any particular subgroup or sequence motif. Furthermore, we have identified another dehydrogenase subgroup with chemoselectivity for imine reduction. Enantioselectivities were determined for the reduction of the model substrate 2-phenylpiperideine, and the effect of changing the reaction conditions was also studied for the reductive aminations of 1-indanone, acetophenone, and 4-methoxyphenylacetone. We have performed sequence-structure analysis to help explain clusters in activity across a phylogenetic tree and to inform rational engineering, which, in one case, has conferred a change in chemoselectivity that had not been previously observed.

Effects of response preference on resistance to change.

Treatments based on differential reinforcement of alternative behavior, such as functional communication training, are widely used. Research regarding the maintenance of related treatment effects is limited. Nevin and Wacker (2013) provided a conceptual framework, rooted in behavioral momentum theory, for the study of treatment maintenance that addressed two components: (a) reemergence of problem behavior, and (b) continued expression of appropriate behavior. In the few studies on this topic, focus has been on variables impacting the reemergence of problem behavior, with fewer studies evaluating the persistence of appropriate behavior. Given the findings from applied research related to functional communication training, variables related to response topography, such as response preference, may impact this aspect of maintenance. In the current study, the impact of response preference on persistence was evaluated in the context of functional communication training for individuals who did not exhibit problem behavior (Experiment 1) and for individuals with a history of reinforcement for problem behavior (Experiment 2). High-preferred mands were more persistent than low-preferred mands. These findings suggest that response related variables, such as response preference, impact response persistence and further suggest that response related variables should be considered when developing interventions such as functional communication training.

Cystic Fibrosis & disorders of the large intestine: DIOS, constipation, and colorectal cancer.

Since 1966 when the Cystic Fibrosis Foundation Patient Registry (CFFPR) was founded, clinicians have witnessed significant advances in both the quality and quantity of life for patients living with Cystic Fibrosis (CF). As patients with CF live longer and fuller lives, increasing encumbrances from gastrointestinal manifestations of CF will be observed. This article serves to discuss "below the diaphragm" concerns involving the large intestine (Distal Intestinal Obstruction Syndrome, Constipation, and Colorectal Cancer). Avenues for development and implementation of clinical care protocols, particularly regarding proactive management of known associated conditions and cancer screening, will continue to be refined in the coming years. It falls to the multidisciplinary CF care team to be actively engaged in addressing these concerns effectively as priority shifts from relative acuity (typically related to early nutrition and lung function) to the travails of longevity as the CF population continues to age.

Three-dimensional segmentation of breast masses from digital breast tomosynthesis images.

Assessment of three-dimensional (3-D) morphology and volume of breast masses is important for cancer diagnosis, staging, and treatment but cannot be derived from conventional mammography. Digital breast tomosynthesis (DBT) provides data from which 3-D mass segmentation could be obtained. Our method combined Gaussian mixture models based on intensity and a texture measure indicative of in-focus structure, gray-level variance. Thresholding these voxel probabilities, weighted by distance to the estimated mass center, gave the final 3-D segmentation. Evaluation used 40 masses annotated twice by a consultant radiologist on in-focus slices in two diagnostic views. Human intraobserver variability was assessed as the overlap between repeated annotations (median 77% and range 25% to 91%). Comparing the segmented mass outline with probability-weighted ground truth from these annotations, median agreement was 68%, and range was 7% to 88%. Annotated and segmented diameters correlated well with histological mass size (both Spearman's rank correlations [Formula: see text]). The volumetric segmentation demonstrated better agreement with tumor volumes estimated from pathology than volume derived from radiological annotations (95% limits of agreement [Formula: see text] to 11 ml and [Formula: see text] to 41 ml, respectively). We conclude that it is feasible to assess 3-D mass morphology and volume from DBT, and the method has the potential to aid breast cancer management.

Preoperative implant selection for unilateral breast reconstruction using 3D imaging with the Microsoft Kinect sensor.

AIMS: This study aimed to investigate whether breast volume measured preoperatively using a Kinect 3D sensor could be used to determine the most appropriate implant size for reconstruction. METHODS: Ten patients underwent 3D imaging before and after unilateral implant-based reconstruction. Imaging used seven configurations, varying patient pose and Kinect location, which were compared regarding suitability for volume measurement. Four methods of defining the breast boundary for automated volume calculation were compared, and repeatability assessed over five repetitions. RESULTS: The most repeatable breast boundary annotation used an ellipse to track the inframammary fold and a plane describing the chest wall (coefficient of repeatability: 70 ml). The most reproducible imaging position comparing pre- and postoperative volume measurement of the healthy breast was achieved for the sitting patient with elevated arms and Kinect centrally positioned (coefficient of repeatability: 141 ml). Optimal implant volume was calculated by correcting used implant volume by the observed postoperative asymmetry. It was possible to predict implant size using a linear model derived from preoperative volume measurement of the healthy breast (coefficient of determination R2 = 0.78, standard error of prediction 120 ml). Mastectomy specimen weight and experienced surgeons' choice showed similar predictive ability (both: R2 = 0.74, standard error: 141/142 ml). A leave one-out validation showed that in 61% of cases, 3D imaging could predict implant volume to within 10%; however for 17% of cases it was >30%. CONCLUSION: This technology has the potential to facilitate reconstruction surgery planning and implant procurement to maximise symmetry after unilateral reconstruction.

Analysis of facial morphology of UK and US general election candidates: Does the 'power face' exist?

AIMS: To determine the frequency of recognised morphologic facial phenotypes among the UK and US election candidates and to assess whether there is a relationship between facial morphology and success in general elections. METHODS: Facial morphology analysis of prime ministerial and presidential candidates in the UK and US elections was performed. Direct facial measurements were made from frontal images and facial morphological indices calculated for all candidates. Anthropometric facial phenotype was determined and comparisons made between the elected leaders and unelected runner-up candidates. Paired candidates who had different facial types were analysed as a subgroup, with the probabilities of electoral success calculated. RESULTS: Data were available for 45 subjects: 22 election winners and 23 unelected runner-ups. Our data showed some variation in facial morphology between the groups. The predominant facial phenotype in both groups was leptoprosopic (long and/or narrow face), accounting for 40% (n = 18). Mesoprosopic (average dimension) and euryprosopic (short and/or broad) facial type represented 31% (n = 14) and 18% (n = 8), respectively. A majority of the sample (n = 36) demonstrated a malar width to mandibular angle width ratio consistent with the modern 'power face' proportion. Subgroup analysis showed a significant association with success in an election when leptoprosopic candidates were paired against candidates of another facial phenotype (p < 0.05). CONCLUSION: Leptoprosopic (long and/or narrow) facial phenotype with a wide mandibular angle width is predominant in the UK and US election candidates. Moreover, this facial phenotype appears to be associated with greater electoral success, supporting the concept of a modern political 'power face'.

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

Evaluation of Kinect 3D Sensor for Healthcare Imaging.

Microsoft Kinect is a three-dimensional (3D) sensor originally designed for gaming that has received growing interest as a cost-effective and safe device for healthcare imaging. Recent applications of Kinect in health monitoring, screening, rehabilitation, assistance systems, and intervention support are reviewed here. The suitability of available technologies for healthcare imaging applications is assessed. The performance of Kinect I, based on structured light technology, is compared with that of the more recent Kinect II, which uses time-of-flight measurement, under conditions relevant to healthcare applications. The accuracy, precision, and resolution of 3D images generated with Kinect I and Kinect II are evaluated using flat cardboard models representing different skin colors (pale, medium, and dark) at distances ranging from 0.5 to 1.2 m and measurement angles of up to 75°. Both sensors demonstrated high accuracy (majority of measurements <2 mm) and precision (mean point to plane error <2 mm) at an average resolution of at least 390 points per cm2. Kinect I is capable of imaging at shorter measurement distances, but Kinect II enables structures angled at over 60° to be evaluated. Kinect II showed significantly higher precision and Kinect I showed significantly higher resolution (both p < 0.001). The choice of object color can influence measurement range and precision. Although Kinect is not a medical imaging device, both sensor generations show performance adequate for a range of healthcare imaging applications. Kinect I is more appropriate for short-range imaging and Kinect II is more appropriate for imaging highly curved surfaces such as the face or breast.

ESPGHAN and NASPGHAN Report on the Assessment of Exocrine Pancreatic Function and Pancreatitis in Children.

The purpose of this clinical report is to discuss several recent advances in assessing exocrine pancreatic insufficiency (EPI) and pancreatitis in children, to review the array of pancreatic function tests, to provide an update on the inherited causes of EPI, with special emphasis on newly available genetic testing, and to review newer methods for evaluating pancreatitis.

Does a patient's Mallampati score predict outcome after maxillomandibular advancement for obstructive sleep apnoea?

The Mallampati airway classification has been used to estimate the success of uvulopalatopharyngoplasty in patients with obstructive sleep apnoea (OSA) but its predictive value in maxillomandibular advancement has not been proved. We aimed to explore the association between preoperative Mallampati scores and surgical outcome after bimaxillary advancement for OSA. We retrospectively analysed data on 50 patients who had maxillofacial operations for OSA at our hospital and stratified them into two groups based on Mallampati scores: high (class III/IV) and low (class I/II). We compared pre- and postoperative apnoea/hypopnoea indices (AHIs), Epworth sleepiness scores, and lowest recorded oxygen saturation in both groups. The postoperative values for all three outcome measures were not significantly different when patients were stratified according to the Mallampati classification (mean (SD) AHI was 41(19) before and 7 (6) after operation in the low group, and 42 (15) before and 9 (7) after in the high group). Success rates (AHI less than 15 postoperatively) were similar in both low and high score groups (p>0.05). Maxillomandibular advancement alleviates obstruction at multiple levels and our study has shown comparable surgical outcomes in both groups. The Mallampati score can be used to optimise patient selection for surgeons considering single-level procedures for OSA. Our study suggests that the Mallampati classification is less useful for the prediction of surgical outcome after maxillomandibular advancement surgery.

Effects of maxillomandibular advancement on systemic blood pressure in patients with obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with resistant hypertension. We investigated to what extent maxillomandibular advancement affected a patient's blood pressure postoperatively. We retrospectively analysed consecutive patients who had Bimaxillary advancement for OSA at our hospital following referral from the local sleep clinic. We collected relevant data on clinical characteristics and explored the changes in systolic and diastolic blood pressures, as well as mean arterial pressure (MAP) preoperatively, with those taken 6 months following surgery. We identified 51 patients with a mean (SD) age of 44 (8) years and a mean (SD) body mass index of 29 (3.4). Preoperative and postoperative data on blood pressure were available for analysis in 45. The mean (SD) systolic blood pressure was significantly reduced in our sample following surgery (from 131(12.6) to 127 (12.5)mmHg, p<0.001). The mean (SD) reduction in postoperative MAP values in the overall group, approached statistical significance (recorded MAP 96.6(10) to 93.1(8)mmHg, p=0.06). In a subgroup of 10 patients who had established hypertension the reduction in values postoperatively (mean reduction: systolic blood pressure 6 mmHg, diastolic blood pressure 10 mmHg, mean arterial pressure 9 mmHg) was greater than that observed in the overall group. Our results have shown an improvement in systemic blood pressure after maxillomandibular advancement for OSA, particularly in those with established hypertension. The data suggest that in addition to being a highly effective treatment for OSA, this surgery may more effectively lower systemic blood pressure than other treatment modalities.

How often does the operating list follow the planned order? An analysis of elective maxillofacial operating lists.

OBJECTIVES: The authors explored consistency of the observed running order in operating sequence compared with prior scheduled listing. We analysed potential variables felt to be predictive in the chances of a patient having their procedure as previously scheduled. METHODS: Data were retrospectively collected for a consecutive group of patients who underwent elective maxillofacial procedures over a four week period. The consistency of scheduled and observed running order was documented. We considered four independent variables (original list position, day of week, morning or afternoon list, seniority of surgeon) and analysed their relationship to the probability of a patient undergoing their operation as per listing. Logistic regression analysis was used to determine significant associations between predictor variables with an altered list order. RESULTS: Data were available for 35 lists (n = 133). 49% of lists were found to run according to prior given order, the remainder subject to some alteration. Logistic regression analysis showed a statistically significant association between original scheduled position and day of week, with list position consistency. Patients listed first were twelve times more likely to have their operation as listed compared to those placed fourth (OR 12.7, 95% CI 3.7-43, p < 0.05). Operating lists at the start of a week were subject to less alteration (p < 0.05). There was no demonstrated relationship between the grade of surgeon operating and alteration in operating sequence. CONCLUSION: Approximately half of lists showed some alteration to the previously printed order. It appears that being first on an elective list offers the greatest guarantee that a patient will have their operation as per prior schedule. It may be reasonable for clinicians to be mindful of potential operating list alterations when preparing their patients for elective surgery.

Equine-associated maxillofacial injuries: retrospective 5-year analysis.

We explored the relation between the causes of facial injuries in equestrians and the presence or absence of associated injuries. Over a 5-year period we retrospectively reviewed all patients who presented to the John Hunter Hospital, New South Wales, with facial injuries that had resulted from activity with horses. We analysed the rates of hard and soft tissue injuries, and of associated injuries by sex and mechanism. A total of 85 patients were included (50 female and 35 male) with an age range of 2-88 years. There was a significant difference in the rate of maxillofacial and associated injuries when groups were analysed for sex and mechanism of injury. Facial injuries caused by falling from a horse were more often associated with other injuries in men than in women (p<0.05), and men were 4 times more likely to present with associated injuries than women (OR 3.9; 95% CI 1.1 to 14) We also found significant differences in the rates of facial fracture. Women who had been kicked by a horse were more likely to sustain bony injuries than men (p<0.05). Our data confirm the association between kicks and facial fracture, and this may provide an impetus for the development of appropriate protective equipment. Patients who sustain facial injuries when falling from a horse often present with associated injuries and this has practical implications for clinicians involved in their management.

The influence of measurement location on reliability of quantitative nailfold videocapillaroscopy in patients with SSc.

OBJECTIVES: Nailfold videocapillaroscopy is being increasingly used as a marker of SSc-related microvascular disease, including in response to treatment. However, it requires further validation. Our aim was to assess the inter-observer, intra-observer and test-retest variability of semi-automated measurement of capillary features as well as of a manual density measurement. METHODS: All capillary apexes in images from 58 patients with SSc were marked up independently by two trained observers (inter-observer variability). The first observer then re-marked the images (intra-observer variability), and finally, the first observer marked up a second image of the same nailfold (test-retest). Mark-up of capillaries was carried out on cropped mosaic images (cropped independently by the observers to a fixed width, to allow the same length of nail bed to be studied for each patient) and on whole mosaic images (examining the whole nail bed). RESULTS: Reproducibility of independently cropped mosaic images was poor and was due to the variation in the positioning of the cropped area. However, quantification of whole mosaic images was highly reproducible, e.g. for inter-capillary distance, the intra-class correlation coefficient for inter-observer, intra-observer and test-retest reliability was 0.95, 0.98 and 0.90 (compared with 0.88, 0.79 and 0.89 for cropped mosaic images), respectively. Intra-observer limits of agreement for whole mosaic images were better than inter-observer reproducibility. CONCLUSION: Quantitative assessment of SSc-related change in nailfold capillaries is unreliable if examination of the same set of capillaries cannot be guaranteed. Conversely a wide-field, high-magnification system that allows visualization of the whole nail bed offers a highly reproducible approach for quantitative assessment and therefore has potential as an outcome measure.

Bilateral breast reduction surgery in elderly women--a retrospective review of outcomes.

BACKGROUND: Breast reduction surgery is successful in improving symptoms associated with breast hypertrophy. We sought to substantiate this in the elderly patient and ascertain how they regarded the outcome. METHODS: The WHO defines elderly as 60 and over. All elderly patients undergoing BBR for breast hypertrophy over a ten-year period at a single institution were identified. Retrospective review of medical records was conducted and patients asked to complete a postal questionnaire. RESULTS: 25/29 (86%) patients responded. 88% felt surgery improved appearance and symptoms. A statistically significant improvement in symptoms, appearance and confidence was demonstrated. 54% of patients voiced negative concerns regarding their surgery. One third were unhappy with final appearance. 2/24 regretted surgery. CONCLUSIONS: Although studies showing positive outcomes for breast reduction in breast hypertrophy are many, none have specifically looked at elderly patients. Most patients seek relief of functional symptoms. Cosmesis seems to be less an issue. That they achieve improvement in symptoms is reassuring, despite longevity of symptoms and possible concomitant musculoskeletal degeneration. However despite satisfaction with relief of symptoms, much dissatisfaction can be felt over less than perfect cosmesis. We feel attention to detail is key, even in the elderly undergoing functional procedures.