Genetically programmed chiral organoborane synthesis.

Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

A prospective study on vulvovaginal candidiasis: multicentre molecular epidemiology of pathogenic yeasts in China.

BACKGROUND: Vulvovaginal candidiasis (VVC) is frequent in women of reproductive age, but very limited data are available on the epidemiology in cases of VVC in China. OBJECTIVES: The current study has been conducted to reveal the prevalence, species distribution of yeast causing VVC and molecular genetics of Candida albicans in China. METHODS: Vaginal swabs were collected from 543 VVC outpatients recruited in 12 hospitals in China between September 2017 and March 2018. They were preliminarily incubated on Sabouraud dextrose agar and then positive subjects of which were then transmitted to our institute for further identification. CHROMagar™ was used to isolate Candida species, and all isolates were finally identified by DNA sequencing. Multilocus sequence typing (MLST) was used to analyse phylogenetic relationships of the various C. albicans isolates. RESULTS: Eleven different yeast species were identified in 543 isolates, among which C. albicans (84.7%) was the most frequent, followed by C. glabrata (8.7%). We obtained 117 unique diploid sequence types from 451 clinical C. albicans isolates and 92 isolates (20.4%) belonged to a New Clade. All the strains appearing in the New Clade were from northern China and they were isolated from non-recurrent VVC. CONCLUSIONS: Our findings suggest that C. albicans are still the main cause of VVC in China and the majority of C. albicans isolates belongs to Clade 1 with DST 79 and DST 45 being two most common. Moreover, the New Clade revealed in our study seems to be specific to northern China.

Machine learning-assisted directed protein evolution with combinatorial libraries.

To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning into the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine-learning models trained on tested variants provide a fast method for testing sequence space computationally. We validated this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (i.e., stereodivergence) of a new-to-nature carbene Si-H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee (enantiomeric excess). By greatly increasing throughput with in silico modeling, machine learning enhances the quality and diversity of sequence solutions for a protein engineering problem.

Origin and Control of Chemoselectivity in Cytochrome c Catalyzed Carbene Transfer into Si-H and N-H bonds.

A cytochrome c heme protein was recently engineered to catalyze the formation of carbon-silicon bonds via carbene insertion into Si-H bonds, a reaction that was not previously known to be catalyzed by a protein. High chemoselectivity toward C-Si bond formation over competing C-N bond formation was achieved, although this trait was not screened for during directed evolution. Using computational and experimental tools, we now establish that activity and chemoselectivity are modulated by conformational dynamics of a protein loop that covers the substrate access to the iron-carbene active species. Mutagenesis of residues computationally predicted to control the loop conformation altered the protein's chemoselectivity from preferred silylation to preferred amination of a substrate containing both N-H and Si-H functionalities. We demonstrate that information on protein structure and conformational dynamics, combined with knowledge of mechanism, leads to understanding of how non-natural and selective chemical transformations can be introduced into the biological world.

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase.

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

Which is the best treatment of osteoporotic vertebral compression fractures: balloon kyphoplasty, percutaneous vertebroplasty, or non-surgical treatment? A Bayesian network meta-analysis.

The aim of the current study was to use a Bayesian network meta-analysis to evaluate the relative benefits and risks of balloon kyphoplasty (BK), percutaneous vertebroplasty (PVP), and non-surgical treatment (NST) for patients with osteoporotic vertebral compression fractures (OVCFs). The results demonstrate that for pain and functional status, PVP was significantly better than NST, while the three treatments did not significantly differ in other outcomes. INTRODUCTION: BK, PVP, and NST are widely used to treat OVCFs, but preferable treatment is unknown. The aim of the current study was to use a Bayesian network meta-analysis to evaluate the relative benefits and risks of BK, PVP, and NST for patients with OVCFs. METHODS: PubMed, EMBASE, and the Cochrane Library were screened. Based on the preplanned eligibility criteria, we screened and included randomized controlled trials that compared BK, PVP, and NST in treating patients with OVCFs. The risk of bias for individual studies was appraised. The data were pooled using a Bayesian network meta-analysis and a traditional direct comparison meta-analysis. RESULTS: Of the 1057 relevant studies, 15 were eligible and included. Compared with NST, PVP significantly reduced pain, Oswestry Disability Index (ODI), and Roland-Morris Disability Questionnaire (RMDQ). The comparative efficacy of BK and PVP was similar for pain (mean difference (MD) 0.51, 95% credible interval (CrI) - 0.35 to 1.4), ODI (MD 0.11, 95% CrI - 13 to 13), and RMDQ (MD 1.2, 95% CrI - 2.7 to 5.4). The European Quality of Life-5 Dimensions (EQ-5D) and Physical Component Summary subscales of the Medical Outcomes Study 36-Item Short-Form General Health Survey (SF-36 PCS) did not differ significantly. There were also no substantial differences in the risks of subsequent vertebral fractures, adjacent vertebral fractures, and re-fractures at the treated level across all comparators. The results of pairwise meta-analyses were almost consistent with those of network meta-analyses. The treatment ranking indicated that PVP had the highest probability of being the most effective for pain, ODI, RMDQ, and EQ-5D. BK had the highest probability of improving SF-36 PCS and of reducing the risk of subsequent vertebral fractures and re-fractures at the treated level. NST was ranked first in preventing adjacent vertebral fractures. CONCLUSION: PVP was the most effective method for improving pain, functional status, and quality of life (based on EQ-5D). BK emerged as the best intervention for decreasing the risk of subsequent vertebral fractures and re-fractures at the treated level. NST could be ranked first in reducing adjacent vertebral fractures. The future directions of OVCFs treatment will depend on the outcomes of additional and larger randomized trials in comparing BK with PVP.

[The importance of therapeutic muds of the Arasan-Kundyzdy deposit for the development of peloid therapy in Kazakhstan]. / Znachenie lechebnykh griazei mestorozhdeniia Arasan-Kundyzdy dlia razvitiia peloidoterapii v Kazakhstane.

BACKGROUND: To provide the population with inexpensive and effective medical products of domestic production is one of the most important public health tasks. These may include therapeutic muds, the high therapeutic value of which is confirmed by many years of positive experience with peloid therapy. The basis for interest in therapeutic muds is their efficiency in treating many diseases and regularly discovered new opportunities for their use. OBJECTIVE: To strive to streamline the use of previously discovered therapeutic muds in Kazakhstan, to search for new hydromineral resources, and to register the latter as minerals. MATERIAL AND METHODS: The material was the results of the analysis of published data on studies of therapeutic muds, their use for medicinal purposes in sanatorium and extrasanatorium conditions, which had been prepared during the grant financing project 'Development of an innovative procedure for the qualitative and quantitative assessment of therapeutic muds in Southeastern Kazakhstan as hydromineral raw materials' (IRN AR 05130934) in 2018. RESULTS AND CONCLUSION: The paper provides a brief description of the results of the study of therapeutic mud deposits in Western Kazakhstan, North Kazakhstan, and the Almaty Region. It gives the main list of diseases in which the use of mud therapy is effective.

Catalyst-Controlled, Enantioselective, and Diastereodivergent Conjugate Addition of Aldehydes to Electron-Deficient Olefins.

A chiral-amine-catalyzed enantioselective and diastereodivergent method for aldehyde addition to electron-deficient olefins is presented. Hydrogen bonding was used as a control element to achieve unusual anti selectivity, which was further elucidated through mechanistic and computational studies.

An Expedient Total Synthesis of Chivosazole†F: an Actin-Binding Antimitotic Macrolide from the Myxobacterium Sorangium Cellulosum.

A unified strategy for the chemical synthesis of the chivosazoles is described. This strategy is based on two closely related approaches involving the late-stage installation of the isomerization-prone (2Z,4E,6Z,8E)-tetraenoate motif, and an expedient fragment-assembly procedure. The result is a highly convergent total synthesis of chivosazole F through the orchestration of three mild Pd/Cu-mediated Stille cross-coupling reactions, including the use of a one-pot, site-selective, three-component process, in combination with controlled installation of the requisite alkene geometry.

Traumatic spinal cord injury in Tianjin, China: a single-center report of 354 cases.

STUDY DESIGN: Hospital-based retrospective study. OBJECTIVES: The objective of this study was to describe the epidemiological profile of traumatic spinal cord injury (TSCI) in Tianjin Medical University General Hospital, China, from 2009 to 2014. SETTING: Tianjin Medical University General Hospital. METHODS: Hospital medical records of patients with TSCI admitted to hospital from 1 January 2009 to 31 December 2014 were reviewed. Collected variables included gender, age, marital status, ethnic group, occupation, etiology, neurological level of injury, American Spinal Injury Association (ASIA)-ISCoS impairment scale at admission, the severity, death and its cause, concomitant injuries and treatment choice. RESULTS: During the study period, 354 cases were identified. Male-to-female ratio was 2.34:1, with a mean age of 50.1±15.5 years. Falls (55.1%), comprising low falls and high falls (33.6% and 21.5%, respectively), were the leading cause, followed by motor vehicle collisions (MVCs) (35.9%). The most common injury site was the cervical spinal cord, especially C4-C6, accounting for 59.3%. Surgery was the major treatment choice (57.6%). CONCLUSION: The number of TSCI patients increased annually in our center. The mean age at the time of injury was older, and the proportion of males was higher. The leading two causes were falls and MVCs. The SCIs caused by MVCs were increasing. Peasants, workers and unemployed individuals were those at higher risk. Surgery was the major treatment choice. These data may be useful to implement those preventive strategies focused on the characteristics of different groups and pay more attention to high-risk populations.

[Mid- to long-term outcomes of cervical disc arthroplasty for symptomatic cervical disc disease: a meta-analysis].

Objective: To compare the benefits and harms of cervical disc arthroplasty (CDA) with anterior cervical discectomy and fusion(ACDF) for symptomatic cervical disc disease at mid- to long-term follow-up. Methods: Electronic searches were made in PubMed, EMBASE, and the Cochrane Library for randomized controlled trials with at least 48 moths follow-up.Outcomes were reported as relative risk or standardized mean difference.Meta-analysis was carried out using Revman version 5.3 and Stata version 12.0. Results: Seven trials were included, involving 2 302 participants.The results of this meta-analysis indicated that CDA brought about fewer secondary surgical procedures, lower neck disability index (NDI) scores, lower neck and arm pain scores, greater SF-36 Physical Component Summary (PCS) and Mental Component Summary(MCS) scores, greater range of motion (ROM) at the operative level and less superior adjacent-segment degeneration(P<0.05) than ACDF.CDA was not statistically different from ACDF in inferior adjacent-segment degeneration, neurological success, and adverse events (P>0.05). Conclusions: CDA can significantly reduce the rates of secondary surgical procedures compared with ACDF.Meanwhile, CDA is superior or equivalent to ACDF in other aspects.As some studies without double-blind are included and some potential biases exites, more randomized controlled trials with high quality are required to get more reliable conclusions.

Leptin-mediated regulation of ICAM-1 is Rho/ROCK dependent and enhances gastric cancer cell migration.

BACKGROUND: Our previous study indicates that leptin enhances gastric cancer (GC) invasion. However, the exact effect of leptin on GC metastasis and its underlying mechanism remain unclear. Intercellular adhesion molecule-1 (ICAM-1), a major molecule in stabilising cell-cell and cell-extracellular matrix interactions, is overexpressed and has crucial roles in tumour metastasis. METHODS: Here, we investigated leptin and ICAM-1 expression in GC tissues. Furthermore, we characterised the influence of leptin on ICAM-1 expression in GC cells and elucidated the underlying mechanism. RESULTS: Leptin and ICAM-1 were overexpressed in GC tissues, and a strong positive correlation was observed. They were also related with clinical stage or lymph node metastasis. Furthermore, leptin induced GC cell (AGS and MKN-45) migration by upregulating ICAM-1, and knockdown of ICAM-1 by small interference RNA (siRNA) blocked this process. Cell surface ICAM-1, as well as soluble ICAM-1 (sICAM-1), was also enhanced by leptin. Moreover, leptin increased ICAM-1 expression through Rho/ROCK pathway, which was attenuated by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated protein kinase (ROCK) (Y-27632). CONCLUSIONS: Our findings indicate that leptin enhances GC cell migration by increasing ICAM-1 through Rho/ROCK pathway, which might provide new insight into the significance of leptin in GC.

Improvement of 7α-methoxycephalosporins production by overexpression of cmcJ and cmcI controlled by promoter ermEp* in Streptomyces clavuligerus.

AIM: This study aimed to improve the efficiency of bioconversion of cephalosporin C to 7α-methoxycephalosporin C. METHODS AND RESULTS: We performed the genetic modulation of the expression level of tailoring genes cmcI and cmcJ, which are responsible for the methoxylation of cephalosporin C, to improve the efficiency of bioconversion in the recombinant strains. The results showed that the coordinate adjustment of the enzyme amounts of CmcI and CmcJ in the host strain by placing the ermEp* promoter in the upstream of both cmcI and cmcJ genes in their expression cassette can improve the efficiency of bioconversion from cephalosporin C to 7α-methoxycephalosporin C. Under the optimized cultivation condition, 2·0 g l(-1) of the substrate cephalosporin C can be transformed into 0·78 g l(-1) of 7α-methoxycephalosporin C by the extract of the recombinant strain Streptomyces clavuligerus DYG3003, in which the transcript ratio of cmcJ/cmcI has been engineered to 1·7 : 1. CONCLUSIONS: Coordinate adjustment of the enzyme amounts of CmcI and CmcJ in the host strain can greatly enhance the efficiency of bioconversion from cephalosporin C (CPC) to 7a-methoxycephalosporin (MCPC). SIGNIFICANCE AND IMPACT OF THE STUDY: As one kind of the most important ß-lactam antibiotics, 7α-methoxycephalosporins show stronger activity against penicillin-resistant bacteria than cephalosporins because of the higher stability of 7α-methoxycephalosporins to ß-lactamase. Our study provides a practical strategy to perform the industrial production of the clinically useful 7α-methoxycephalosporin C through biotransformation.

The impact of the Mukaiyama aldol reaction in total synthesis.

Four decades since Mukaiyama's first reports on the successful application of silicon and boron enolates in directed aldol reactions, the ability of this highly controlled carbon-carbon bond-forming method to simultaneously define stereochemistry, introduce complexity, and construct the carbon skeleton with a characteristic 1,3-oxygenation pattern has made it a powerful tool for natural product synthesis. This Minireview highlights a number of representative total syntheses that demonstrate the impact of the Mukaiyama aldol reaction and discusses the underlying mechanistic rationale that determines the stereochemical outcomes.

Up-regulation of HLA-G expression in cervical premalignant and malignant lesions.

The human leukocyte antigen G (HLA-G) molecule, a non-classical major histocompatibility complex class I antigen, exhibits highly limited tissue distribution and gene variation. Recent studies indicate strong immunoinhibitory properties in tumor cells that may favor their escape from anti-tumor immune responses. However, the role of HLA-G in cervical premalignant and malignant lesions has not been defined clearly. In our study, HLA-G expression was studied in cervical tissue from 119 patients with lesions and 22 normal cervical tissue specimens by immunohistochemistry. HLA-G was expressed in 45% (54/119) of cervical lesion-containing tissues while it was rarely detectable (0/22) in the control specimens (P = 0.000). ROC curve analysis showed that HLA-G has an area under the curve (AUC) of 0.694. Furthermore, we investigated soluble HLA-G expression in the plasma of 172 patients with cervical lesions and 20 healthy controls. Significant increases were also observed in soluble HLA-G levels (median, 191.4 vs 45.18 U/ml, P < 0.001). The relative operating characteristic (ROC) curves for soluble HLA-G (sHLA-G), squamous cell carcinoma (SCC), and carbohydrate antigen 125 (CA125) show an AUC of 0.710, 0.634, and 0.588, respectively. At the cut-off values of 108.20 U/ml for sHLA-G, 1.5 ng/ml for SCC, and 35 U/ml for CA125, the sensitivity was 73.30%, 47.83%, and 44.83%, respectively. The detection of soluble HLA-G in plasma may have significance in the early detection of cervical malignant lesions.

Changes in serum S100A12 and sRAGE associated with improvement of the PaO(2)/FiO(2) ratio following PMX-DHP therapy for postoperative septic shock.

BACKGROUND: Endotoxin (Et) adsorption therapy with a column of polymyxin B-immobilized fibers (PMX) is effective in improving the partial pressure of arterial oxygen/fraction of inspired oxygen ratio (PaO(2)/FiO(2) ratio) and increasing mean arterial blood pressure (MAP) in sepsis. S100A12 and soluble receptor for advanced glycation end product (sRAGE) are useful as early markers of acute lung injury. PURPOSE: To investigate the effect of improving the PaO(2)/FiO(2) ratio by PMX-direct hemoperfusion (PMX-DHP) on production of S100A12 and sRAGE. SUBJECTS AND METHODS: Sepsis patients after surgery for perforation of the lower gastrointestinal tract were adopted as the subjects. We retrospectively reviewed the cases of 20 patients on mechanical ventilation and continuous administration of norepinephrine. We recorded PaO(2)/FiO(2) ratio, MAP, and norepinephrine doses. S100A12, sRAGE, and Et levels were measured before and after PMX-DHP. RESULTS: The PaO(2)/FiO(2) ratio and MAP improved significantly after PMX-DHP (p < 0.05). S100A12 and Et decreased significantly after PMX-DHP (p < 0.05). No differences were observed in sRAGE. CONCLUSION: S100A12 is useful as a marker that reflected improvement in the PaO(2)/FiO(2) ratio after PMX-DHP. We consider PMX-DHP to be useful as adjunctive therapy for sepsis that reduces the Et and corrects the pathology in the early stage.

Cancer incidence in British Indians and British whites in Leicester, 2001-2006.

BACKGROUND: Incidence rates for many cancers are lower in India than in Britain and it is therefore of interest to compare rates in British Indians to British whites, as well as to rates in India. We present estimates for Leicester, which has the largest population of Indian origin in Britain, and also has virtually complete, self-assigned, ethnicity data. METHODS: We obtained data on all cancer registrations from 2001 to 2006 for Leicester with ethnicity data obtained by linkage to the Hospital Episode Statistics database. Age-standardised incidence rates were calculated for British Indians and British whites as well as incidence rate ratios, adjusted for age and income. RESULTS: Incidence rate ratios for British Indians compared with British whites were significantly less than 1.0 for all cancers combined (0.65) and for cancer of the breast (0.72), prostate (0.76), colon (0.46), lung (0.30), kidney (0.36), stomach (0.54), bladder (0.48) and oesophagus (0.64), but higher than 1.0 for liver cancer (1.95). CONCLUSION: These results are likely to be the most accurate estimate of cancer incidence in British Indians to date and confirm that cancer incidence in British Indians is lower than in British whites in Leicester, particularly for cancer of the breast, prostate, colon and lung (and other smoking-related cancers), but much higher than in India.

Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida.

Stable infection of Bacillus anthracis laboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two such B. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines of Eisenia fetida earthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group of Bacillus species and related organisms, Wip1 and Wip4 were both infective only toward B. anthracis and certain B. cereus strains. The natural host of Wip4 remained constant for 3 years and was a B. cereus strain that expressed a B. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which such B. cereus- and B. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability of B. cereus sensu lato to form heat-resistant spores, the sensitivity of B. anthracis to the PlyG lysin, or the selective amplification of environmental phages cocultured with B. anthracis. Findings from this work indicate that B. cereus sensu lato and its phages are common inhabitants of earthworm intestines.

Genetic screening of 202 individuals with congenital limb malformations and requiring reconstructive surgery.

BACKGROUND: Congenital limb malformations (CLMs) are common and present to a variety of specialties, notably plastic and orthopaedic surgeons, and clinical geneticists. The authors aimed to characterise causative mutations in an unselected cohort of patients with CLMs requiring reconstructive surgery. METHODS: 202 patients presenting with CLM were recruited. The authors obtained G-banded karyotypes and screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for point mutations using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. Multiplex ligation dependent probe amplification (MLPA) kits were developed and used to measure copy number in GLI3, HOXD13, ROR2, SALL1, SALL4, TBX5 and the ZRS of SHH. RESULTS: Within the cohort, causative genetic alterations were identified in 23 patients (11%): mutations in GLI3 (n = 5), HOXD13 (n = 5), the ZRS of SHH (n = 4), and chromosome abnormalities (n = 4) were the most common lesions found. Clinical features that predicted the discovery of a genetic cause included a bilateral malformation, positive family history, and having increasing numbers of limbs affected (all p<0.01). Additionally, specific patterns of malformation predicted mutations in specific genes. CONCLUSIONS: Based on higher mutation prevalence the authors propose that GLI3, HOXD13 and the ZRS of SHH should be prioritised for introduction into molecular genetic testing programmes for CLM. The authors have developed simple criteria that can refine the selection of patients by surgeons for referral to clinical geneticists. The cohort also represents an excellent resource to test for mutations in novel candidate genes.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.