The Role of Telemedicine in Strabismus Assessment: A Narrative Review and Meta-Analysis.

Purpose: Strabismus is a common ocular condition requiring precise quantification of gaze deviation and qualification of strabismus category. Telemedicine refers to the use of technology to remotely diagnose and treat medical conditions. This narrative review aimed to assess the efficacy of a variety of telemedicine modalities for the assessment of strabismus. A secondary objective was to quantify overall accuracy, sensitivity, and specificity of automated methods using meta-analysis of available data. Methods: A literature search was conducted using the Ovid MEDLINE, Embase, and Cochrane Library data libraries. Keywords, including "strabismus," "phoria," "telemed*," and "telehealth," were used to locate relevant studies, with Medical Subject Headings terms, free text, and synonyms. No year restrictions were applied. Studies not in English were excluded. Risk of bias was assessed using the QUADAS-2 tool. Results: Thirty-four studies were included. All outcomes relating to accuracy and reliability of telemedicine versus a reference standard were extracted, as well as qualitative observations. High sensitivity, specificity, accuracy, and agreement were consistently shown across studies. Meta-analysis of two subsets featuring automated methods, for which relevant data were available, revealed a pooled accuracy of 0.877 (0.806-0.949), sensitivity of 0.856 (0.805-0.907), and specificity of 0.900 (0.845-0.954). Subcategories "remote standard assessment," "digital image analysis," "wearable devices," "mobile health (mHealth)," and "artificial intelligence" were independently examined. Conclusions: The majority of systems achieved parity with standard physician assessment, with the added benefit of eliminating subjectivity. Meta-analysis results suggest potential introduction of remote automated assessment where conventional assessment is unavailable, although accuracy of current technologies remains limited compared to in-person examination. Telemedicine modalities described offer convenience for patients, shorter examination times, and the potential to go beyond in-person assessments. The evidence gathered in this review supports the beginning of telemedicine integration into the world of strabismus diagnosis.

Temporal-to-Nasal Macular Ganglion Cell and Inner Plexiform Layer Ratios in a Large Adult Twin Cohort: Correlations With Age and Heritability.

Purpose: Temporal-to-nasal macular ganglion cell layer thickness ratios are reduced in albinism. We explored similar ratios in a large twin cohort to investigate ranges in healthy adults, correlations with age, and heritability. Methods: More than 1000 twin pairs from TwinsUK underwent macular optical coherence tomography (OCT) scans. Automated segmentation yielded thicknesses for the combined ganglion cell and inner plexiform layer (GCIPL) in Early Treatment of Diabetic Retinopathy Study subfields. Participants with diseases likely to affect these layers or segmentation accuracy were excluded. Inner and outer ratios were defined as the ratio of temporal-to-nasal GCIPL thickness for inner and outer subfields respectively. Corresponding ratios were obtained from a smaller cohort undergoing OCTs with a different device (three-dimensional (3D)-OCT, Topcon, Japan). Results: Scans from 2300 twins (1150 pairs) were included (mean [SD] age, 53.9 (16.5) years). Mean (SD) inner and outer ratios were 0.89 (0.09) and 0.84 (0.11), correlating negatively with age (coefficients, -0.17 and -0.21, respectively). In males (150 pairs) ratios were higher and did not correlate significantly with age. Intrapair correlation coefficients were higher in monozygotic than dizygotic pairs; age-adjusted heritability estimates were 0.20 and 0.23 for inner and outer ratios, respectively. For the second cohort (n = 166), mean (SD) ratios were 0.93 (0.08) and 0.91 (0.09), significantly greater than for the larger cohort. Conclusions: Our study gives reference values for temporal-to-nasal macular GCIPL subfield ratios. Weak negative correlations with age emerged. Genetic factors may contribute to ∼20% to 23% of the variance in healthy individuals. The ratios differ according to the OCT platform used.

Systematic review and meta-analyses on the accuracy of diagnostic tests for IgE-mediated food allergy.

The European Academy of Allergy and Clinical Immunology (EAACI) is updating the Guidelines on Food Allergy Diagnosis. We aimed to undertake a systematic review of the literature with meta-analyses to assess the accuracy of diagnostic tests for IgE-mediated food allergy. We searched three databases (Cochrane CENTRAL (Trials), MEDLINE (OVID) and Embase (OVID)) for diagnostic test accuracy studies published between 1 October 2012 and 30 June 2021 according to a previously published protocol (CRD42021259186). We independently screened abstracts, extracted data from full texts and assessed risk of bias with QUADRAS 2 tool in duplicate. Meta-analyses were undertaken for food-test combinations for which three or more studies were available. A total of 149 studies comprising 24,489 patients met the inclusion criteria and they were generally heterogeneous. 60.4% of studies were in children ≤12 years of age, 54.3% were undertaken in Europe, ≥95% were conducted in a specialized paediatric or allergy clinical setting and all included oral food challenge in at least a percentage of enrolled patients, in 21.5% double-blind placebo-controlled food challenges. Skin prick test (SPT) with fresh cow's milk and raw egg had high sensitivity (90% and 94%) for milk and cooked egg allergies. Specific IgE (sIgE) to individual components had high specificity: Ara h 2-sIgE had 92%, Cor a 14-sIgE 95%, Ana o 3-sIgE 94%, casein-sIgE 93%, ovomucoid-sIgE 92/91% for the diagnosis of peanut, hazelnut, cashew, cow's milk and raw/cooked egg allergies, respectively. The basophil activation test (BAT) was highly specific for the diagnosis of peanut (90%) and sesame (93%) allergies. In conclusion, SPT and specific IgE to extracts had high sensitivity whereas specific IgE to components and BAT had high specificity to support the diagnosis of individual food allergies.

Prevalence, Concordance, and Heritability of Vitreomacular Interface Abnormalities in a Twin Study.

Purpose: The relative importance of genetic factors in common vitreomacular interface (VMI) abnormalities is unknown. The aim of this classical twin study is to determine the prevalence case wise concordance between monozygotic and dizygotic twin pairs, and heritability of common VMI abnormalities, including epiretinal membrane (ERM), posterior vitreous detachment (PVD), vitreomacular adhesion (VMA), vitreomacular traction (VMT), lamellar macular holes (LMHs), and full-thickness macular holes (FTMHs). Methods: This is a single-center, cross-sectional classical twin study of 3406 TwinsUK participants over the age of 40 years who underwent spectral domain macular optical coherence tomography (SD-OCT) scans which were graded for signs of VMI abnormalities. Case wise concordance was calculated and the heritability of each VMI abnormality was estimated using OpenMx structural equation modeling. Results: In this population (mean age = 62.0 years [SD = 10.4 years], range = 40-89 years) the overall prevalence of ERM was 15.6% (95% confidence interval [CI] = 14.4-16.9) and increased with age, posterior vitreous detachment affected 21.3% (20.0-22.7), and VMA was diagnosed in 11.8% (10.8-13.0). Monozygotic twins were more concordant for all traits than dizygotic twins, and age, spherical equivalent refraction (SER), and lens status-adjusted heritability was estimated at 38.9% (95% CI = 33.6-52.8) for ERM, 53.2% (95% CI = 41.8-63.2) for PVD, and 48.1% (95% CI = 33.6-58) for VMA. Conclusions: Common VMI abnormalities are heritable and therefore have an underlying genetic component. Given the sight-threatening potential of VMI abnormalities, further genetic studies, such as genomewide association studies, would be useful to identify genes and pathways implicated in their pathogenesis.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Metagenomic discovery of feruloyl esterases from rumen microflora.

Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow's rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1633 to 4143 bp, and the ORFs from 681 to 1359 bp. BLAST search reveals sequence homology to feruloyl esterases and esterases/lipases identified in anaerobes. The seven genes were expressed in Escherichia coli, and the proteins were purified to homogeneity. The FAEs were found to cover types B, C, and D in the feruloyl esterase classification system using model hydroxycinnamic acid esters. The release of ferulic acid (FA) catalyzed by these enzymes was established using natural substrates corn fiber (CF) and wheat insoluble arabinoxylan (WIA). Three of the enzymes were demonstrated to cleave diferulates and hence the capability to break down Araf-FA-FA-Araf cross-links. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.

A novel method for rapid and sensitive metagenomic activity screening.

Direct cloning of metagenomes has proven to be a powerful tool for the exploration of the diverse sequence space of a microbial community leading to gene discovery and biocatalyst development. The key to such approach is the development of rapid, sensitive, and reliable functional screening of libraries. The majority of library screen have relied on the use of agar plates in petri dishes incorporating the target enzyme substrate for activity detection of positive clones (Iqbal et al. [1], Knietsch et al. [2], Popovic et al. [3]). In this article, a novel method is described consisting of: (1) formulation and application of substrate gel microtiter assay plates, (2) screening of libraries of clones in split pools in the wells of the assay plate, and (3) progressive enrichment and isolation of individual positive clones. The method has been successfully used in the rapid discovery of novel genes and enzymes from rumen microbial metagenome with high efficacy. •Novel substrate gel assay plates for activity screening with localized and intensified signals.•Rapid and complete screening of library clones in split pools.•Progressive enrichment scheme as a refining step for isolating target gene.

The Anne Boleyn Illusion is a Six-Fingered Salute to Sensory Remapping.

The Anne Boleyn Illusion exploits the somatotopic representation of touch to create the illusion of an extra digit and demonstrates the instantaneous remapping of relative touch location into body-based coordinates through visuo-tactile integration. Performed successfully on thousands, it is also a simple demonstration of the flexibility of body representations for use at public events, in schools or in the home and can be implemented anywhere by anyone with a mirror and some degree of bimanual coordination.

Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan.

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present.

Cloning of a Novel Feruloyl Esterase from Rumen Microbial Metagenome for Substantial Yield of Mono- and Diferulic Acids from Natural Substrates.

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone released ferulic acid (FA) and diferulic acid (diFA) from wheat insoluble arabinoxylan (WIA) and other natural substrates. The diFA released was confirmed by mass spectrometry. A maximum of 205±5.7 µg FA and 0.84±0.1 µg diFA were released (37°C, pH 6.5, 2 hr) when a saturating amount of RuFae4 (23 nmole for 100 mg WIA) was used. These yields represent 48.3% of FA, and 6.6% of diFAs present in the WIA substrate. Addition of GH10 endoxylanase (EX) to RuFae4 both at 1 nmole concentrations increased the release of FA and diFAs by 17 and 10 fold, respectively. Addition of GH11 EX resulted in smaller increase in the amount of both FA and diFAs. Applying additive amount of the two enzymes did not lead to additive increase in the product yields, suggesting that it was primarily the GH10 enzyme contributing synergism to FA/diFA release in mixed reactions.

Structural basis for xyloglucan specificity and α-d-Xylp(1 → 6)-D-Glcp recognition at the -1 subsite within the GH5 family.

GH5 is one of the largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the crystal structures of two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 XEGs from parental cellulases and a new mode of action within the GH5 family related to structural adaptations in the -1 subsite. The oligosaccharide found in the XEG5A complex, permitted the mapping, for the first time, of the positive subsites of a GH5 XEG, revealing the importance of the pocket-like topology of the +1 subsite in conferring the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the negative subsites, completing the subsite mapping of GH5 XEGs at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl side chain at this position. The stereochemical compatibility of the -1 subsite with a xylosyl moiety was also reported for other structurally nonrelated XEGs belonging to the GH74 family, indicating it to be an essential attribute for this mode of action.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases.

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 µg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate.

Comparative characterization of a bifunctional endo-1,4- ß-mannanase/1,3-1,4- ß-glucanase and its individual domains.

A fusion gene isolated from a microbial metagenome encodes a N-terminal endo-1,4- ß-mannanase and a C-terminal 1,3-1,4- ß -glucanase,. The full-length gene and the individual N- and C-domains were separately cloned and expressed in E coli. The purified whole enzyme hydrolyzed glucomannan, galactomannan, and ß-glucan with Km and kcat values 2.2, 2.6, 3.6 mg/ml, and 302, 130, 337 min -1 , respectively. The hydrolysis of ß-glucan by the C domain enzyme decreased significantly with added glucomannan to the reaction, suggesting inhibition effect. Analogous result was not observed with the N domain enzyme when ß-glucan was added to the reaction. The whole enzyme did not show improvement of efficiency compared to the individual or additive total hydrolysis of the two domain enzymes using single or mixed substrates.

Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity.

A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA(3)Xyl(3)), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-ß-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-ß-1,4-glucanase EG I. XYN IV was able to degrade several different ß-1,4-xylans, but was inactive on ß-1,4-mannans and ß-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble ß-1,3-ß-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward ß-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.

Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme.

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 µM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-ß-D-xylopyranoside as the substrate.

Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass.

The Aspergillus niger feruloyl esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium with a yield of ~2 mg/l. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interaction chromatography. The specific activity was determined to be 8,200 U/µg (pH 6.5, 20°C, 3.5 mM 4-nitrophenyl ferulate). The protein had a correct N-terminal sequence of ASTQGISEDLY, indicating that the signal peptide was properly processed. The FAE exhibited an optimum pH of 6-7 and operated optimally at 50°C using ground switchgrass as the substrate. The yeast clone was demonstrated to catalyze the release of ferulic acid continuously from switchgrass in YNB medium at 30°C. This work represents the first report on engineering yeast for the breakdown of ferulic acid crosslink to facilitate consolidated bioprocessing.

Cloning and characterization of an exo-xylogucanase from rumenal microbial metagenome.

A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.

Improved survival in red blood cell transfusion dependent patients with primary myelofibrosis (PMF) receiving iron chelation therapy.

Many patients with primary myelofibrosis (PMF) become red blood cell (RBC) transfusion dependent (TD), risking iron overload (IOL). Iron chelation therapy (ICT) may decrease the risk of haemosiderosis associated organ dysfunction, though its benefit in PMF is undefined. To assess the effect of TD and ICT on survival in PMF, we retrospectively reviewed 41 patients. Clinical data were collected from the database and by chart review. The median age at PMF diagnosis was 64 (range 43-86) years. Median white blood cell (WBC) count at diagnosis was 7.6 (range 1.2-70.9) x 10(9)/L; haemoglobin 104 (62-145) G/L; platelets 300 (38-2088) x 10(9)/L. Lille, Strasser, Mayo and International Prognostic System (IPS) scores were: low risk, n = 15, 8, 11, 3; intermediate, n = 15, 19, 9, 16; high, n = 5, 11, 5, 7; respectively. Primary PMF treatment was: supportive care, n = 23; hydroxyurea, n = 10; immunomodulatory, n = 4; splenectomy, n = 2. Sixteen patients were RBC transfusion independent (TI) and 25 TD; of these 10 received ICT for a median of 18.3 (0.1-117) months. Pre-ICT ferritin levels were a median of 2318 (range 263-8400) and at follow up 1571 (1005-3211 microg/L (p = 0.01). In an analysis of TD patients, factors significant for overall survival (OS) were: WBC count at diagnosis (p = 0.002); monocyte count (p = 0.0001); Mayo score (p = 0.05); IPS (p = 0.02); number of RBC units (NRBCU) transfused (p = 0.02) and ICT (p = 0.003). In a multivariate analysis, significant factors were: NRBCU (p = 0.001) and ICT (p = 0.0001). Five year OS for TI, TD-ICT and TD-NO ICT were: 100, 89 and 34%, respectively (p = 0.003). The hazard ratio (HR) for receiving >20 RBCU was 7.6 (95% Confidence Intervals [CI] 1.2-49.3) and for ICT was 0.15 (0.03-0.77). In conclusion, 61% of PMF patients developed RBC-TD which portended inferior OS; however patients receiving ICT had comparatively improved OS, suggesting a clinical benefit. Prospective studies of IOL and the impact of ICT in PMF are warranted.

A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies.

A novel xyloglucan-specific endo-beta-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of approximately 90 amino acid residues with unknown function. The catalytic domain assumed an (alpha/beta)(8)-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis-Menten kinetics, yielding K (m) and V (max) of 3.61 +/- 0.23 mg/ml and 0.30 +/- 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K (i) of 1.46 +/- 0.13 mM. The enzyme was devoid of transglycosylase activities.