Superior survival of ex vivo cultured human reticulocytes following transfusion into mice.

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

The development and implementation of the SWEET Peer Review Programme for pediatric diabetes centres.

This paper describes the background to the development, implementation, and subsequent management of a programme of peer review of Pediatric Diabetes Centres belonging to the SWEET Group The paper summarizes the overall purpose of the programme, lists the principles upon which the programme is founded, and sets out the intended outcomes of the programme Details are given of the way in which the programme is delivered and summarizes the key findings from the 16 centres reviewed to date Finally the paper highlights the feedback that has been received from those who have been reviewed and those who have acted as reviewers and discusses ways in which the programme can be further developed in the future.

Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis.

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34(+) cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis. Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ß1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A-decorated vesicles of autophagic origin.

Arthroscopic resection of talocalcaneal coalitions--a bicentre case series of a new technique.

BACKGROUND: Symptomatic tarsal coalitions failing conservative treatment are traditionally managed by open resection. We describe an arthroscopic technique for excising talocalcaneal coalitions and present a retrospective two-surgeon case series of the first eight patients (nine feet). METHODS: Outcome measures include restoration of subtalar movements, return to work and sports, visual analogue pain scales and Sports Athlete Foot and Ankle Scores (SAFAS). Follow-up ranges from 1 to 5.5 years. RESULTS: Pain and SAFAS improved in 7 patients. Subtalar movements were improved in all feet. All patients achieved early good function but one relapsed requiring subsequent fusions. One posterior tibial nerve was damaged. CONCLUSIONS: Minimal destruction of bone and soft tissues allows early mobilization and minimizes pain. Patient selection and preoperative planning are crucial to avoid relapse and complication. This series from two independent surgeons supports the feasibility and effectiveness of this technique.

Recombinant production of self-assembling ß-structured peptides using SUMO as a fusion partner.

BACKGROUND: Self-assembling peptides that form nanostructured hydrogels are important biomaterials for tissue engineering scaffolds. The P11-family of peptides includes, P11-4 (QQRFEWEFEQQ) and the complementary peptides P11-13 (EQEFEWEFEQE) and P11-14 (QQOrnFOrnWOrnFOrnQQ). These form self-supporting hydrogels under physiological conditions (pH 7.4, 140 mM NaCl) either alone (P11-4) or when mixed (P11-13 and P11-14). We report a SUMO-peptide expression strategy suitable for allowing release of native sequence peptide by SUMO protease cleavage. RESULTS: We have expressed SUMO-peptide fusion proteins from pET vectors by using autoinduction methods. Immobilised metal affinity chromatography was used to purify the fusion protein, followed by SUMO protease cleavage in water to release the peptides, which were recovered by reverse phase HPLC. The peptide samples were analysed by electrospray mass spectrometry and self-assembly was followed by circular dichroism and transmission electron microscopy. CONCLUSIONS: The fusion proteins were produced in high yields and the ß-structured peptides were efficiently released by SUMO protease resulting in peptides with no additional amino acid residues and with recoveries of 46% to 99%. The peptides behaved essentially the same as chemically synthesised and previously characterised recombinant peptides in self-assembly and biophysical assays.

Arthroscopic resection of calcaneonavicular coalition/malunion via a modified sinus tarsi approach: an early case series.

BACKGROUND: Calcaneonavicular coalition represents abnormal coalescence between calcaneus and navicular bone. It is a congenital anomaly, sometimes becoming symptomatic in young adolescent. This is managed conservatively initially, failing which surgical excision, open or arthroscopic, is considered. We present our arthroscopic technique via a modified sinus tarsi approach, with early results in two adolescent and two young adult patients. METHOD: The patient is placed in a 45° "saggy" lateral position, and entry points for portals are marked around sinus tarsi area. The adequacy of resection is checked with image intensifier at the end of procedure. Patients complete subjective scoring forms, Manchester-Oxford Foot Questionnaire and Visual Analogue Scale, pre operatively and at follow-ups. Paired t test was performed to assess statistical significance. RESULTS: The results of early follow-up of these patients have confirmed complete excision, non-recurrence and symptomatic improvement. The mean difference in MOXFQ scores pre and post surgery is 39.33, with a two-tailed p value of 0.0187. Similarly, the mean difference in VAS score is 5.67 with a two-tailed p value of 0.0034. These are statistically significant and confirm symptomatic improvement at an early follow-up. CONCLUSION: The arthroscopic technique provides better access allowing wide excision and causes minimal soft tissue trauma leading to early recovery and mobilisation.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Correction and prevention of deformity in type II tibialis posterior dysfunction.

UNLABELLED: Cobb described a method of reconstruction in Johnson and Strom Type II tibialis posterior dysfunction (TPD) using a split tibialis anterior musculotendinous graft. We assessed patient function and satisfaction after a modified Cobb reconstruction in a group of patients with a narrow spectrum of dysfunction, examined a modification of the Johnson and Strom classification to emphasize severity of deformity, and assessed the ability of the technique to prevent subsequent fixed deformity. We prospectively followed 32 patients managed by this technique and a translational os calcis osteotomy with early flexible deformity after failed conservative treatment. There were 28 women and four men with unilateral disease. The average followup was 5.1 years. Staging was confirmed clinically and with imaging. The modified surgery involved a bone tunnel in the navicular rather than the medial cuneiform with plaster for 8 weeks followed by orthotics and physiotherapy. All of the osteotomies healed and 29 of the 32 patients could perform a single heel rise test at 12 months. The mean postoperative American Orthopaedic Foot and Ankle Society hindfoot score was 89. One patient had a superficial wound infection and one a temporary dysesthesia of the medial plantar nerve; both resolved. The observations suggest the technique is a comparable method of treating early Johnson and Strom Type II TPD. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Endoscopic Resection of Tarsal Coalitions.

Endoscopic resection of tarsal coalitions is technically feasible for both talocalcaneal and calcaneonavicular coalitions. Careful consideration of each individual patient is necessary before proceeding with endoscopic resection. Endoscopic resection of these coalitions may offer benefits in terms of faster recovery and less wound problems, but this has not been proven. Several case reports and case series appear in the literature and are reviewed here along with the different techniques reported. Better-quality evidence is required to assess the clinically relevant benefits and the recurrence rate for endoscopic resection in comparison with open resection.

The Genomic Landscape of Intrinsic and Acquired Resistance to Cyclin-Dependent Kinase 4/6 Inhibitors in Patients with Hormone Receptor-Positive Metastatic Breast Cancer.

Mechanisms driving resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in hormone receptor-positive (HR+) breast cancer have not been clearly defined. Whole-exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of estrogen receptor expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Three of these activating alterations-in AKT1, RAS, and AURKA-have not, to our knowledge, been previously demonstrated as mechanisms of resistance to CDK4/6i in breast cancer preclinically or in patient samples. Together, these eight mechanisms were present in 66% of resistant tumors profiled and may define therapeutic opportunities in patients. SIGNIFICANCE: We identified eight distinct mechanisms of resistance to CDK4/6i present in 66% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ metastatic breast cancer.This article is highlighted in the In This Issue feature, p. 1079.

Effects of Two Competitive Soccer Matches on Landing Biomechanics in Female Division I Soccer Players.

Fatigue has been proposed to increase the risk of knee injury. This study tracked countermovement jump, knee isometric strength, and kinetics and kinematics in 8 female soccer players (experimental group) during an anticipated sidestep maneuver before and after two matches played over a 43-h period. Time points were: Before and after match 1 (T0 and T1), 12 h after the first match (T2), and immediately after the second match (T3). A control group participated only in practice sessions. Isometric knee extension strength decreased by 14.8% at T2 (p = 0.003), but knee flexion was not affected until T3, declining by 12.6% (p = 0.018). During the sidestep maneuver, knee joint degrees of flexion at initial contact was increased by 17.1% at T3, but maximum knee and hip angle at initial contact were unchanged. Peak resultant ground reaction force (GRF) increased by 12.6% (p = 0.047) at T3 (3.03 xBW) from 2.69 xBW at T0, while posterior GRF was significantly higher than T0 at all three subsequent time points (T1 = 0.82 ± 0.23 xBW, T2 = 0.87 ± 0.22 xBW, T3 = 0.89 ± 0.22 xBW). Anterior tibial shear force increased significantly (p = 0.020) at T3 (1.24 ± 0.12 xBW) compared to T1 (1.15 ± 0.13 xBW), an 8.8% increase. Lateral tibial shear force was significantly higher at both T1 (0.95 ± 0.20 xBW) and T3 (1.15 ± 0.38 xBW) compared to T0 (0.67 ± 0.25 xBW). These findings suggest that participation in a soccer match has significant effects on both physical performance parameters and kinetics/kinematics during a sidestep cut, but these can be more pronounced after a second match with short rest.

From invisibility to readability: Recovering the ink of Herculaneum.

The noninvasive digital restoration of ancient texts written in carbon black ink and hidden inside artifacts has proven elusive, even with advanced imaging techniques like x-ray-based micro-computed tomography (micro-CT). This paper identifies a crucial mistaken assumption: that micro-CT data fails to capture any information representing the presence of carbon ink. Instead, we show new experiments indicating a subtle but detectable signature from carbon ink in micro-CT. We demonstrate a new computational approach that captures, enhances, and makes visible the characteristic signature created by carbon ink in micro-CT. This previously "unseen" evidence of carbon inks, which can now successfully be made visible, is a discovery that can lead directly to the noninvasive digital recovery of the lost texts of Herculaneum.

Aurora A Kinase Inhibition Is Synthetic Lethal with Loss of the RB1 Tumor Suppressor Gene.

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

New mutations in C1GALT1C1 in individuals with Tn positive phenotype.

Tn polyagglutination results from inactivating mutations in C1GALT1C1, an X-borne gene encoding a core 1 beta3-galactosyltransferase-specific molecular chaperone (cosmc) required for the functioning of T-synthase (beta 1,3-galactosyltransferase), a glycosyltransferase essential for the correct biosynthesis of O-glycans. This study found novel inactivating mutations (Glu152Lys, Ser193Pro and Met1Ile) in the coding sequence of C1GALT1C1 in three Tn positive individuals and a complete lack of C1GALT1C1 cDNA expression was observed in an additional Tn positive individual. In addition, expression of ST6GALNAC1, which encodes (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 and gives rise to sialyl-Tn antigen, was present at comparable levels in normal and Tn-positive human erythroblasts. Expression studies of wild-type and Tn positive C1GALT1C1 cDNA in the Jurkat cell line confirmed that the amino acid substitutions observed in Tn are inactivating. Analysis of the transcriptome of cultured normal and Tn positive erythroblasts revealed numerous differences in gene expression. Reduced transcript levels for fatty acid binding protein 5 (FABP5) and plexin D1 (PLXND1), and increased levels for aquaporin 3 (AQP3) were confirmed by quantitative real-time polymerase chain reaction. These data show that alteration of O-glycan structures resulting from T-synthase deficiency is accompanied by altered expression of a wide variety of genes in erythroid cells.

Benchmarking of a Simple Scintigraphic Test for Gastro-oesophageal Reflux Disease That Assesses Oesophageal Disease and Its Pulmonary Complications

Objectives: Gastro-oesophageal reflux disease (GORD) is both common and troubling with a prevalence of 20-40%. We assessed the utility of a scintigraphic reflux study to evaluate the oesophageal and extra-oesophageal manifestation of disease compared to the standard tests such as pH monitoring and manometry. Methods: Patients were recruited into a prospective database of referrals to a tertiary referral center for either resistance to maximal medical therapy or extra-oesophageal symptoms of GORD. Data included 2 channel 24-hour pH monitoring and manometry results, as well as scintigraphic reflux data with late images assessing pulmonary aspiration of refluxate. Results: Study population included 250 patients (155 F, 95 M) with an average age of 60 years. Patients were clinically classified as either GORD (n=72) or laryngopharyngeal reflux (LPR) (n=178). Pulmonary aspiration of the refluxate was detected significantly more commonly in LPR patients (58/178 compared with GORD 10/72). Strong correlations were found between the scintigraphic time-activity curves in the upper oesophagus and pharynx, and ineffective oesophageal motility and pulmonary aspiration. pH studies correlated with the scintigraphic studies but did not predict aspiration similar to other modalities when evaluated by ROC analysis. Conclusion: Scintigraphic reflux studies offer a viable alternative test for GORD and extra-oesophageal manifestations of reflux disease. Strong correlations were found between measurable scintigraphic parameters and oesophageal motility and lung aspiration of refluxate. This may provide a more confident decision analysis in patients being considered for fundoplication for troubling extra-oesophageal symptoms.

Percutaneous screw fixation for painful non-union of lateral malleolus ankle fractures.

INTRODUCTION: Lateral malleolus non-union can cause pain and loss of function. Standard treatment involves open approach with debridement, bone-grafting and plate stabilisation, with published surgical site infection rates to 17%. To minimise the risk of soft tissue complications and allow early mobilisation, we describe a technique for percutaneous cannulated screw stabilisation. MATERIALS AND METHODS: Retrospective case review for all percutaneous lateral malleolus non-union stabilisation procedures undertaken in our hospital between 2011 and 2017 was performed. Fracture union was diagnosed by resolution of pain and swelling, with a return to full weight-bearing mobilisation and two-view radiographs consistent with union. RESULTS: Twelve cases were reviewed. All fractures united. There was one superficial wound infection treated with oral antibiotics, and one early case with drill-piece fracture requiring conversion to open procedure with plate stabilisation. CONCLUSION: We believe this to be the first report of percutaneous stabilisation for non-union of lateral malleolus fractures. We demonstrate this to be a safe and effective technique.

Arthroscopic ankle arthrodesis.

BACKGROUND: Arthroscopic arthrodesis has been used mainly for in situ fusion of arthritic ankles without deformity. This paper presents the application of arthroscopic arthrodesis of ankles with marked deformity. METHODS: The results of 78 consecutive cases of arthroscopic ankle arthrodeses, performed in 74 patients, were retrospectively evaluated. Forty-eight ankles had minor deformity (group A), whereas 30 ankles had a varus or valgus deformity of more than 15 degrees (maximum 45 degrees) (group B). The average hospital stay was 3.8 and 3.4 days in groups A and B, respectively (p = 0.74). Postoperative treatment included ankle immobilization for 3 months. Progressive weightbearing was initiated at 2 weeks. Mean followup was 21.1 months. RESULTS: Fusion occurred in 47 of 48 (97.9%) ankles in group A at an average time of 13.1 +/- 5.8 weeks and in 29 of 30 (96.7%) ankles at 11.6 +/- 2.4 weeks in group B (p = 0.19). Unplanned operative procedures were required in 11 ankles (14.1%). One superficial wound infection occurred. Symptomatic arthritis from the adjacent joints developed in six ankles (7.7%). Postoperative ankle alignment in the frontal plane averaged 0.7 and 0.4 degrees of valgus (p = 0.41), whereas the sagittal plane angle averaged 106 +/- 4 degrees and 104.5 +/- 7 degrees in groups A and B, respectively (p = 0.22). The outcome was graded as very good in 79.2% (38 feet) in group A and 80% (24 feet) in group B, fair in 18.8% (9 feet) in group A and 16.7% (5 feet) in group B and poor in one ankle in each group (p = 0.68). CONCLUSIONS: The arthroscopic technique offered high fusion rates and low morbidity. Deformity correction was achieved with good results.

Comparison of posttonsillectomy pain using the ultrasonic scalpel, coblator, and electrocautery.

OBJECTIVE: To compare postoperative tonsillectomy pain between 3 commonly used surgical devices: the Harmonic Ultrasonic Scalpel (Ethicon Endo-Surgery, Cincinnati, OH), the Coblator (ArthroCare Corp, Sunnyvale, CA), and electrocautery. STUDY DESIGN AND SETTING: A prospective, randomized trial. One hundred thirty-four patients were randomly assigned to receive a tonsillectomy with 1 of 3 surgical devices. All patients were asked to fill out a postoperative diary. RESULTS: Statistically significant differences in pain scores were revealed between the Coblator and electrocautery (P = 0.02) and between the Coblator and the Ultrasonic Scalpel (P = 0.003), with the Coblator having lower pain scores. Electrocautery and the Ultrasonic Scalpel did not differ significantly from each other. The Coblation method showed a strong trend toward quicker return to normal diet. CONCLUSION: Patients undergoing tonsillectomy with the Coblator device reported less pain over a 10-day period than patients undergoing tonsillectomy with electrocautery or the Ultrasonic Scalpel. Pain after tonsillectomy remains a major issue for our patients. The choice of surgical instrument appears to be one way to reduce this pain. EBM RATING: A-1b.