Hydrolysis of (1,4)-beta-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-beta-D-mannan endohydrolase and beta-D-mannosidase

A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.     

Landscape spatial configuration is a key driver of wild bee demographics

The majority of studies investigating the effects of landscape composition and configuration on bee populations have been conducted in regions of intensive agricultural production, ignoring regions which are dominated by seminatural habitats, such as the islands of the Aegean Archipelago. In addition, research so far has focused on the landscape impacts on bees sampled in cropped fields while the landscape effects on bees inhabiting seminatural habitats are understudied. Here, we investigate the impact of the landscape on wild bee assemblages in 66 phryganic (low scrubland) communities on 8 Aegean islands. We computed landscape metrics (total area and total perimeter–area ratio) in 4 concentric circles (250, 500, 750, and 1000 m) around the center of each bee sampling site including 3 habitat groups (namely phrygana, cultivated land, and natural forests). We further measured the local flower cover in 25 quadrats distributed randomly at the center of each sampling site. We found that the landscape scale is more important than the local scale in shaping abundance and species richness of bees. Furthermore, habitat configuration was more important than the total area of habitats, probably because it affects bees’ movement across the landscape. Phrygana and natural forests had a positive effect on bee demographics, while cultivated land had a negative effect. This demonstrates that phryganic specialists drive bee assemblages in these seminatural landscapes. This finding, together with the shown importance of landscape scale, should be considered for the management of wild bees with special emphasis placed on the spatial configuration of seminatural habitats.     

How Outpatient Palliative Care Teleconsultation Facilitates Empathic Patient-Professional Relationships: A Qualitative Study

Objective

The problems and needs of advanced cancer patients and proxies normally increase as the disease progresses. Home-based advanced cancer patients and their proxies benefit from collaborations between primary care physicians and hospital-based palliative care specialists when confronted with complex problems in the last phase of life. Telemedicine might facilitate direct, patient-centered communication between patients and proxies, primary care physicians, and specialist palliative care teams (SPCTs). This study focuses on the impact of teleconsultation technologies on the relationships between home-based palliative care patients and hospital-based palliative care specialists.

Methods

This work consists of a qualitative study among patients, family members, and caregivers that utilizes long-term direct observations, semi-structured interviews, and open interviews following the observations.

Results

The analysis of the empirical data resulted in three key concepts that describe the impact of teleconsultation on the patient-professional relationship in palliative homecare: transcending the institutional walls of home and hospital; transparency of teleconsultation technology; and technologized, intimate patient-professional relationships. Teleconsultation offers (1) condensed encounters between home-based palliative care patients and distant professionals, (2) a unique insight into the patients’ daily lives for palliative care specialists, and (3) long-term interaction that results in trustful relationships and experiences of intimacy and relief.

Conclusions

Teleconsultation fits the practice of home-based palliative care. Teleconsultation can, if well applied, facilitate computer-mediated but empathic patient-palliative care specialist relationships, which enable professional care attuned to the patient’s context as well as patient involvement. This article proposes a teleconsultation implementation guide for optimal use of teleconsultation in daily palliative care practice.     

Influence of the Accuracy of Angiography-Based Reconstructions on Velocity and Wall Shear Stress Computations in Coronary Bifurcations: A Phantom Study

Introduction

Wall shear stress (WSS) plays a key role in the onset and progression of atherosclerosis in human coronary arteries. Especially sites with low and oscillating WSS near bifurcations have a higher propensity to develop atherosclerosis. WSS computations in coronary bifurcations can be performed in angiography-based 3D reconstructions. It is essential to evaluate how reconstruction errors influence WSS computations in mildly-diseased coronary bifurcations. In mildly-diseased lesions WSS could potentially provide more insight in plaque progression.

Materials Methods

Four Plexiglas phantom models of coronary bifurcations were imaged with bi-plane angiography. The lumens were segmented by two clinically experienced readers. Based on the segmentations 3D models were generated. This resulted in three models per phantom: one gold-standard from the phantom model itself, and one from each reader. Steady-state and transient simulations were performed with computational fluid dynamics to compute the WSS. A similarity index and a noninferiority test were used to compare the WSS in the phantoms and their reconstructions. The margin for this test was based on the resolution constraints of angiography.

Results

The reconstruction errors were similar to previously reported data; in seven out of eight reconstructions less than 0.10 mm. WSS in the regions proximal and far distal of the stenosis showed a good agreement. However, the low WSS areas directly distal of the stenosis showed some disagreement between the phantoms and the readers. This was due to small deviations in the reconstruction of the stenosis that caused differences in the resulting jet, and consequently the size and location of the low WSS area.

Discussion

This study showed that WSS can accurately be computed within angiography-based 3D reconstructions of coronary arteries with early stage atherosclerosis. Qualitatively, there was a good agreement between the phantoms and the readers. Quantitatively, the low WSS regions directly distal to the stenosis were sensitive to small reconstruction errors.     

De novo MECP2 duplications in two females with intellectual disability and unfavorable complete skewed X-inactivation

Xq28 microduplications of MECP2 are a prominent cause of a severe syndromic form of intellectual disability (ID) in males. Females are usually unaffected through near to complete X-inactivation of the aberrant X chromosome (skewing). In rare cases, affected females have been described due to random X-inactivation. Here, we report on two female patients carrying de novo MECP2 microduplications on their fully active X chromosomes. Both patients present with ID and additional clinical features. Mono-allelic expression confirmed complete skewing of X-inactivation. Consequently, significantly enhanced MECP2 mRNA levels were observed. We hypothesize that the cause for the complete skewing is due to a more harmful mutation on the other X chromosome, thereby forcing the MECP2 duplication to become active. However, we could not unequivocally identify such a second mutation by array-CGH or exome sequencing. Our data underline that, like in males, increased MECP2 dosage in females can contribute to ID too, which should be taken into account in diagnostics.     

An experimental test of the role of environmental temperature variability on ectotherm molecular, physiological and life-history traits: implications for global warming

Global climate change is one of the greatest threats to biodiversity; one of the most important effects is the increase in the mean earth surface temperature. However, another but poorly studied main characteristic of global change appears to be an increase in temperature variability. Most of the current analyses of global change have focused on mean values, paying less attention to the role of the fluctuations of environmental variables. We experimentally tested the effects of environmental temperature variability on characteristics associated to the fitness (body mass balance, growth rate, and survival), metabolic rate (VCO(2)) and molecular traits (heat shock protein expression, Hsp70), in an ectotherm, the terrestrial woodlouse Porcellio laevis. Our general hypotheses are that higher values of thermal amplitude may directly affect life-history traits, increasing metabolic cost and stress responses. At first, results supported our hypotheses showing a diversity of responses among characters to the experimental thermal treatments. We emphasize that knowledge about the cellular and physiological mechanisms by which animals cope with environmental changes is essential to understand the impact of mean climatic change and variability. Also, we consider that the studies that only incorporate only mean temperatures to predict the life-history, ecological and evolutionary impact of global temperature changes present important problems to predict the diversity of responses of the organism. This is because the analysis ignores the complexity and details of the molecular and physiological processes by which animals cope with environmental variability, as well as the life-history and demographic consequences of such variability.     

Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Background

Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications.Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia.The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation.

Methods

Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered.

Results

Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p =?0.22), erythrocyte GALT activity (p =?0.87), urinary galactose (p =?0.52) and urinary galactitol levels (p =?0.41). Patients’ fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect.

Conclusions

This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

     

New rodents (Cricetidae) from the Neogene of Curaçao and Bonaire,Dutch Antilles

Cordimus, a new genus of cricetid rodent, is described from Neogene deposits on the islands of Curaçao and Bonaire, Dutch Antilles. The genus is characterized by strongly cuspidate molars, the presence of mesolophs in most upper molars and the absence of mesolophids in lower molars. Similarities with the early cricetid Copemys from the Miocene of North America coupled with apparent derived characters shared with the subfamily Sigmodontinae suggest that Cordimus may be close to the root of the sigmodontine lineage, a possibility that remains to be tested through explicit phylogenetic analysis. Three species are recognized on the basis of size and details of molar morphology. Cordimus hooijeri sp. nov. is described from Bonaire on the basis of Holocene owl pellet material that consists of dentaries and postcranial material only. This species is presumed to be extinct, but focused surveys are needed to confirm this hypothesis. Cordimus debuisonjei sp. nov. and Cordimus raton sp. nov. are described from deposits on Tafelberg Santa Barbara in Curaçao. Although the age of these deposits is not known, they are most likely of late Pliocene or early Pleistocene age. Both are represented by numerous isolated molars and some osteological material.