Evaluation of in silico pathogenicity prediction tools for the classification of small in-frame indels.

BACKGROUND: The use of in silico pathogenicity predictions as evidence when interpreting genetic variants is widely accepted as part of standard variant classification guidelines. Although numerous algorithms have been developed and evaluated for classifying missense variants, in-frame insertions/deletions (indels) have been much less well studied. METHODS: We created a dataset of 3964 small (< 100 bp) indels predicted to result in in-frame amino acid insertions or deletions using data from gnomAD v3.1 (minor allele frequency of 1-5%), ClinVar and the Deciphering Developmental Disorders (DDD) study. We used this dataset to evaluate the performance of nine pathogenicity predictor tools: CADD, CAPICE, FATHMM-indel, MutPred-Indel, MutationTaster2021, PROVEAN, SIFT-indel, VEST-indel and VVP. RESULTS: Our dataset consisted of 2224 benign/likely benign and 1740 pathogenic/likely pathogenic variants from gnomAD (n = 809), ClinVar (n = 2882) and, DDD (n = 273). We were able to generate scores across all tools for 91% of the variants, with areas under the ROC curve (AUC) of 0.81-0.96 based on the published recommended thresholds. To avoid biases caused by inclusion of our dataset in the tools' training data, we also evaluated just DDD variants not present in either gnomAD or ClinVar (70 pathogenic and 81 benign). Using this subset, the AUC of all tools decreased substantially to 0.64-0.87. Several of the tools performed similarly however, VEST-indel had the highest AUCs of 0.93 (full dataset) and 0.87 (DDD subset). CONCLUSIONS: Algorithms designed for predicting the pathogenicity of in-frame indels perform well enough to aid clinical variant classification in a similar manner to missense prediction tools.

Associated thoracic injury in patients with a clavicle fracture: a retrospective analysis of 1461 polytrauma patients.

PURPOSE: During primary survey the main goal is to ascertain life-threatening injuries. A chest X-ray is recommended in all polytrauma patients as thoracic injury plays an important role in mortality. However, treatment-dictating injuries are often missed on the chest X-ray. In contrast, clavicle fractures should be relatively easy to diagnose on a chest X-ray. We previously showed that clavicle fractures occur in approximately 10 % of all polytrauma patients in our population. The aim was to compare polytrauma patients, with and without a clavicle fracture, to investigate if a clavicle fracture is associated with concomitant thoracic injury. METHODS: A retrospective cohort study of polytrauma patients (ISS ≥ 16) from 2007 until 2011. Thoracic injuries were defined as: ribfracture, pneumothorax, lung contusion, sternum fracture, hemothorax, myocardial contusion, thoracic aorta injury and thoracic spine injury. RESULTS: Of 1461 polytrauma patients in 160 patients a clavicle fracture was diagnosed, and 95 % was diagnosed on chest X-ray. Patients with a clavicle fracture had a higher mean Injury Severity Score (ISS) (29.2 ± 10.1 vs. 24.9 ± 9.1; P < 0.001). Additional thoracic injuries were more prevalent in patients with a clavicle fracture (76 vs. 47 %; OR 3.6; 95 % CI 2.45-5.24) and they had a higher rate of thoracic injury with an AIS ≥ 3 (66 vs. 41 %; OR 2.8; 95 % CI 1.97-3.93). CONCLUSIONS: The clavicle can be seen as the gatekeeper of the thorax. In polytrauma patients, a clavicle fracture is easily diagnosed during primary survey and may indicate underlying thoracic injury, as the rate and extent of concomitant thoracic injury are high.

Induced seismicity response of hydraulic fracturing: results of a multidisciplinary monitoring at the Wysin site, Poland.

Shale oil and gas exploitation by hydraulic fracturing experienced a strong development worldwide over the last years, accompanied by a substantial increase of related induced seismicity, either consequence of fracturing or wastewater injection. In Europe, unconventional hydrocarbon resources remain underdeveloped and their exploitation controversial. In UK, fracturing operations were stopped after the Mw 2.3 Blackpool induced earthquake; in Poland, operations were halted in 2017 due to adverse oil market conditions. One of the last operated well at Wysin, Poland, was monitored independently in the framework of the EU project SHEER, through a multidisciplinary system including seismic, water and air quality monitoring. The hybrid seismic network combines surface mini-arrays, broadband and shallow borehole sensors. This paper summarizes the outcomes of the seismological analysis of these data. Shallow artificial seismic noise sources were detected and located at the wellhead active during the fracturing stages. Local microseismicity was also detected, located and characterised, culminating in two events of Mw 1.0 and 0.5, occurring days after the stimulation in the vicinity of the operational well, but at very shallow depths. A sharp methane peak was detected ~19 hours after the Mw 0.5 event. No correlation was observed between injected volumes, seismicity and groundwater parameters.

Getting the feel of food structure with atomic force microscopy.

This article describes the progress in the development of the atomic force microscope as an imaging tool and a force transducer, with particular reference to applications in food science. Use as an imaging tool has matured and emphasis is placed on the novel insights gained from the use of the technique to study food macromolecules and food colloids, and the subsequent applications of this new knowledge in food science. Use as a force transducer is still emerging and greater emphasis is given on the methodology and analysis. Where available, applications of force measurements between molecules or between larger colloidal particles are discussed, where they have led to new insights or solved problems related to food science. The future prospects of the technique in imaging or through force measurements are discussed.

Nanomechanics of Pectin-Linked ß-Lactoglobulin Nanofibril Bundles.

Nanofibrils of ß-lactoglobulin can be assembled into bundles by site-specific noncovalent cross-linking with high-methoxyl pectin (Hettiarachchi et al. Soft Matter 2016, 12, 756). Here we characterized the nanomechanical properties of bundles using atomic force microscopy and force spectroscopy. Bundles had Gaussian cross sections and a mean height of 17.4 ± 1.4 nm. Persistence lengths were calculated using image analysis with the mean-squared end-to-end model. The relationship between the persistence length and the thickness had exponents of 1.69-2.30, which is consistent with previous reports for other fibril types. In force spectroscopy experiments, the bundles stretched in a qualitatively different manner to fibrils, and some of the force curves were consistent with peeling fibrils away from bundles. The flexibility of pectin-linked nanofibril bundles is likely to be tunable by modulating the stiffness and length of fibrils and the ratio of pectin to fibrils, giving rise to a wide range of structures and functionalities.

Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe-host interactions.

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique ß-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host-microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Predicting suitability of intramedullary fixation for displaced midshaft clavicle fractures.

PURPOSE: Implant-related irritation is a technique-specific complication seen in a substantial number of patients treated with intramedullary nailing for clavicle fractures. The purpose of this study was to identify predictors for developing implant-related irritation in patients with displaced midshaft clavicle fractures treated with elastic stable intramedullary nailing. METHODS: A retrospective analysis of the surgical database in two level 2 trauma centers was performed. Patients who underwent intramedullary nailing for displaced midshaft clavicle fractures between 2005 and 2012 in the first hospital were included. Age, gender, fracture comminution and fracture location were assessed as possible predictors for developing irritation using multivariate logistic regression analysis. These predictors were externally validated using data of patients treated in another hospital. RESULTS: Eighty-one patients were included in initial analysis. In the multivariate analysis, comminuted fractures in comparison to non-comminuted fractures (72 vs. 38%, p = 0.027) and fracture location (p < 0.001) were significantly associated with the development of implant-related irritation. In particular, lateral diaphyseal fractures caused irritation compared to fractures on the medial side of the cut-off point (88 vs. 26%). External validation of these predictors in 48 additional patients treated in another hospital showed a similar predictive value of the model and a good fit. CONCLUSION: Comminuted and lateral diaphyseal fractures were found to be statistically significant and independent predictors for developing implant-related irritation. We, therefore, believe that intramedullary nailing might not be suitable for these types of fractures. Future studies are needed to determine whether alternative surgical techniques or implants would be more suitable for these specific types of fractures.

Structural changes in cell wall pectins during strawberry fruit development.

Strawberry (Fragaria × anannasa Duch.) is one of the most important soft fruit. Rapid loss of firmness occurs during the ripening process, resulting in a short shelf life and high economic losses. To get insight into the role of pectin matrix in the softening process, cell walls from strawberry fruit at two developmental stages, unripe-green and ripe-red, were extracted and sequentially fractionated with different solvents to obtain fractions enriched in a specific component. The yield of cell wall material as well as the per fresh weight contents of the different fractions decreased in ripe fruit. The largest reduction was observed in the pectic fractions extracted with a chelating agent (trans-1,2- diaminocyclohexane-N,N,N'N'-tetraacetic acid, CDTA fraction) and those covalently bound to the wall (extracted with Na2CO3). Uronic acid content of these two fractions also decreased significantly during ripening, but the amount of soluble pectins extracted with phenol:acetic acid:water (PAW) and water increased in ripe fruit. Fourier transform infrared spectroscopy of the different fractions showed that the degree of esterification decreased in CDTA pectins but increased in soluble fractions at ripen stage. The chromatographic analysis of pectin fractions by gel filtration revealed that CDTA, water and, mainly PAW polyuronides were depolymerised in ripe fruit. By contrast, the size of Na2CO3 pectins was not modified. The nanostructural characteristics of CDTA and Na2CO3 pectins were analysed by atomic force microscopy (AFM). Isolated pectic chains present in the CDTA fractions were significantly longer and more branched in samples from green fruit than those from red fruit. No differences in contour length were observed in Na2CO3 strands between samples of both stages. However, the percentage of branched chains decreased from 19.7% in unripe samples to 3.4% in ripe fruit. The number of pectin aggregates was higher in green fruit samples of both fractions. These results show that the nanostructural complexity of pectins present in CDTA and Na2CO3 fractions diminishes during fruit development, and this correlates with the solubilisation of pectins and the softening of the fruit.

Unravelling the nanostructure of strawberry fruit pectins by endo-polygalacturonase digestion and atomic force microscopy.

Pectins analysed by AFM are visualized as individual chains, branched or unbranched, and aggregates. To investigate the nature of these structures, sodium carbonate soluble pectins from strawberry fruits were digested with endo-polygalacturonase M2 from Aspergillus aculeatus and visualized by AFM. A gradual decrease in the length of chains was observed as result of the treatment, reaching a minimum LN value of 22nm. The branches were not visible after 2h of enzymatic incubation. The size of complexes also diminished significantly with the enzymatic digestion. A treatment to hydrolyse rhamnogalacturonan II borate diester bonds neither affected chains length or branching nor complex size but reduced the density of aggregates. These results suggest that chains are formed by a mixture of homogalacturonan and more complex molecules composed by a homogalacturonan unit linked to an endo-PG resistant unit. Homogalacturonan is a structural component of the complexes and rhamnogalacturonan II could be involved in their formation.

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins.

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell-surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB-MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

Visualisation of xanthan conformation by atomic force microscopy.

Direct visual evidence obtained by atomic force microscopy demonstrates that when xanthan is adsorbed from aqueous solution onto the heterogeneously charged substrate mica, its helical conformation is distorted. Following adsorption it requires annealing for several hours to restore its ordered helical state. Once the helix state reforms, the AFM images obtained showed clear resolution of the periodicity with a value of 4.7nm consistent with the previously predicted models. In addition, the images also reveal evidence that the helix is formed by a double strand, a clarification of an ambiguity of the xanthan ultrastructure that has been outstanding for many years.

Health-related quality of life in trauma patients who sustained a calcaneal fracture.

BACKGROUND: Calcaneal fractures are known to cause a considerable long-term disability; disability influences the public health negatively in terms of personal suffering and monetary losses. Health-related quality of life (HRQoL) in general is influenced by various patient-specific factors, and possibly trauma and fracture characteristics. Previous studies might have underestimated the impact of this injury because of several injury and patient specific exclusion criteria. In this study we provide an overview of the patient characteristics and outcome of patients with a calcaneal fracture in a Dutch level I trauma population. METHODS: We have performed a retrospective cohort study in a trauma level 1 centre. All patients who sustained a calcaneal fracture and were 16 years or older at time of admission were included. We have retrieved the relevant patient and fracture characteristics from the medical status and evaluated current health status with a questionnaire. We have used the EQ-6D for quantification of the HRQoL. Moreover, the patient's capability to work was evaluated. RESULTS: 125 patients with 151 calcaneal fractures were identified of which 93 patients with 114 calcaneal fractures participated in this study. The median EQ-5D index value is 0.78. All dimensions of the EQ-6D are affected in particular the dimensions 'mobility', 'pain/discomfort' and 'usual activity'. 85% is capable to work. Female patients or patients with a relevant comorbidity or a psychiatric history have a significantly lower HRQoL score. In this study population no difference in HRQoL results was demonstrated among different socio-economic status, associated injuries, or severity of injury. CONCLUSIONS: This study demonstrates that patients who sustained a calcaneal fracture have a significantly lower HRQoL than the Dutch reference population and suffer from a chronic disability. Moreover, patients that have a comorbidity, a relevant psychiatric history or are female have significantly lower HRQoL scores. Furthermore, this study shows that socio-economic status, associated injuries, or severity of injury did not influence the HRQoL in this study population. LEVEL OF EVIDENCE: Prognostic level II.

Chemical characterization of hydrothermally pretreated and enzyme-digested wheat straw: An evaluation of recalcitrance.

There is great interest in understanding changes that occur to cell wall constituents during saccharification of pretreated lignocellulose, particularly in relation to recalcitrance of the residues. This paper reports the effects of hydrothermal pretreatment followed by enzyme hydrolysis on the extractability and properties of recalcitrant wheat straw polymers. The results show that the undigested residue had lost much of its archestructure. Compositional analysis portrayed a considerable loss of cross-linking di-ferulic acid phenolics, hemicellulosic and cellulosic sugars. The remaining cellulosic and non-cellulosic polysaccharides were much more readily extractable in alkali and molecular profiling revealed the presence of low Mw oligomers in the fractions suggesting the partial enzyme hydrolysis of hemicelluloses and cellulose. Simultaneous saccharification and fermentation of the pretreated and enzyme-digested residues surprisingly resulted in ethanol yields of up to 99% of the theoretical. This is discussed in relation to the "recalcitrant" nature of the original pretreated and enzyme digested biomass.

A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA.

We report the discovery and characterization of a double-stranded RNA (dsRNA) mycovirus isolated from the human pathogenic fungus Aspergillus fumigatus, Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1), which reveals several unique features not found previously in positive-strand RNA viruses, including the fact that it represents the first dsRNA (to our knowledge) that is not only infectious as a purified entity but also as a naked dsRNA. The AfuTmV-1 genome consists of four capped dsRNAs, the largest of which encodes an RNA-dependent RNA polymerase (RdRP) containing a unique GDNQ motif normally characteristic of negative-strand RNA viruses. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase capping enzyme and the smallest dsRNA a P-A-S-rich protein that apparently coats but does not encapsidate the viral genome as visualized by atomic force microscopy. A combination of a capping enzyme with a picorna-like RdRP in the AfuTmV-1 genome is a striking case of chimerism and the first example (to our knowledge) of such a phenomenon. AfuTmV-1 appears to be intermediate between dsRNA and positive-strand ssRNA viruses, as well as between encapsidated and capsidless RNA viruses.

Visualization of internal structure of banana starch granule through AFM.

Atomic force microscopy (AFM) is a high resolution technique for studying the external and internal structures of starch granules. For this purpose granules were isolated from bananas and embedded in a non-penetrating resin. To achieve image contrast of the ultrastructure, the face of the cut blocks were wetted in steam and force modulation mode imaging was used. Images of starch from green bananas showed large variation of height across the granule due to a locational specific absorption of water and swelling of amorphous regions; the data reveal that the center of the granules are structurally different and have different viscoelastic properties. Images of starches from ripe bananas showed an even greater different level of organization: absence of growth rings around the hilum; the central region of the granule is richer in amylose; very porous surface with round shaped dark structures; the size of blocklets are larger than the green fruits.

Patient-reported health-related quality of life after a displaced intra-articular calcaneal fracture: a systematic review.

BACKGROUND: A displaced intra-articular calcaneal fracture (DIACF) is known for having a negative influence on the daily activities of patients. A health-related quality of life (HRQoL) outcome instrument is used to quantify the impact of DIACF. It seems that these studies used restrictive inclusion criteria and observe specific patient groups; consequently, an increased risk of bias that results in incorrect estimation of the impact. Therefore, we will systematically review the current literature. MATERIALS AND METHODS: A systematic search was performed in PubMed, Embase and Cochrane library. Inclusion criteria were studies reporting DIACF and HRQoL, measured with SF-36, SF-36v2, EQ-5D or EQ-6D. The identified articles were critically appraised for their relevance and validity. The overall risk of bias was determined. The studies with a low to medium risk of bias were used for data extraction. RESULTS: 32 articles were available for the critical appraisal. 13 articles had a medium risk of bias. All studies reported the SF-36 and two studies also reported the EQ-5D. CONCLUSIONS: This systematic review indicates that DIACF is a life-changing event for most patients. The HRQoL is substantially lower in comparison to the period before the trauma and to the general population, in particular the subdomains related to the physical domain are affected. In addition, this review reveals that the identified studies have a medium to high risk of bias. Consequently, it is challenging to make reliable and valid conclusions. Therefore, we provided recommendations to decrease the risk of bias in order to improve future research.

An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium.

Intestinal crypt fission is a homeostatic phenomenon, observable in healthy adult mucosa, but which also plays a pathological role as the main mode of growth of some intestinal polyps. Building on our previous individual based model for the small intestinal crypt and on in vitro cultured intestinal organoids, we here model crypt fission as a budding process based on fluid mechanics at the individual cell level and extrapolated predictions for growth of the intestinal epithelium. Budding was always observed in regions of organoids with abundant Paneth cells. Our data support a model in which buds are biomechanically initiated by single stem cells surrounded by Paneth cells which exhibit greater resistance to viscoelastic deformation, a hypothesis supported by atomic force measurements of single cells. Time intervals between consecutive budding events, as simulated by the model and observed in vitro, were 2.84 and 2.62 days, respectively. Predicted cell dynamics was unaffected within the original crypt which retained its full capability of providing cells to the epithelium throughout fission. Mitotic pressure in simulated primary crypts forced upward migration of buds, which simultaneously grew into new protruding crypts at a rate equal to 1.03 days(-1) in simulations and 0.99 days(-1) in cultured organoids. Simulated crypts reached their final size in 4.6 days, and required 6.2 days to migrate to the top of the primary crypt. The growth of the secondary crypt is independent of its migration along the original crypt. Assuming unrestricted crypt fission and multiple budding events, a maximal growth rate of the intestinal epithelium of 0.10 days(-1) is predicted and thus approximately 22 days are required for a 10-fold increase of polyp size. These predictions are in agreement with the time reported to develop macroscopic adenomas in mice after loss of Apc in intestinal stem cells.

The adsorption-desorption behaviour and structure function relationships of bile salts.

The digestion of dietary components in the human gastrointestinal (GI) tract is a complex, dynamic, inherently heterogeneous process. A key aspect of the digestion of lipid in the GI tract is the combined action of bile salts, lipase and colipase in hydrolysing and solubilising dispersed lipid. The bile salts are a mixture of steroid acid conjugates with surfactant properties. In order to examine whether the different bile salts have different interfacial properties their dynamic interfacial behaviour was characterised. Differences in the adsorption behaviour to solid hydrophobic surfaces of bile salt species were studied using dual polarisation interferometry and atomic force microscopy (AFM) under physiological conditions. Specifically, the cholates adsorbed more slowly and a significant proportion were irreversibly adsorbed following buffer rinsing; whereas the deoxycholates and chenodeoxycholates adsorbed more rapidly and desorbed to a greater extent following buffer rinsing. The conjugating groups (taurine, glycine) did not influence the behaviour. AFM showed that the interfacial structures that remained following buffer rinsing were also different between these two groups. In addition, the adsorption-desorption behaviour affected the adsorption of colipase to a solid surface. This supports the idea that cooperative adsorption occurs between certain bile salts and colipase to facilitate the adsorption and activity of pancreatic lipase in order to restore lipolytic activity in the presence of bile salts. This study provides insights into how differences in bile salt structure could affect lipase activity and solubilisation of lipolysis products and other lipid-soluble bioactive molecules.

Trehalose-containing hydrocolloid edible films prepared in the presence of transglutaminase.

In this article, edible hydrocolloid films were prepared by using Citrus pectins and the protein phaseolin in the presence of microbial transglutaminase, an enzyme able to catalyze isopeptide bonds between endo-protein-reactive glutamine and lysine residues. For the first time, trehalose, a nonreducing homodisaccharide into which two glucose units are linked together by a α-1,1-glycosidic linkage, was used as a component of hydrocolloid films constituted of both proteins and carbohydrates. Our data have demonstrated that these films act as very effective barriers to gases, especially to CO2 . They also present a high antioxidant capability as measured by the 2,2-diphenyl-1-picrylhydrazyl scavenging assay. In addition, the films were characterized using Atomic Force Microscopy, a powerful tool used to evaluate film surface topography and roughness. The results of our experiments clearly indicate that the trehalose-containing films prepared both in the presence and absence of transglutaminase are composed of nanoparticles with a smooth surface, having similar roughness values (Rα). In conclusion, according to barrier and antioxidant properties and to their structure, it is possible to consider the trehalose-containing films as innovative bioplastics potentially able to protect different kinds of foods.