Multiple unit gastroretentive drug delivery systems: a new preparation method for low density microparticles.
The aim of this study was to develop a new preparation method for low density foam-based, floating microparticles and to demonstrate the systems' performance in vitro. Major advantages of the novel preparation technique include: (i) short processing times, (ii) no exposure of the ingredients to high temperatures, (iii) the possibility to avoid toxic organic solvents, and (iv) high encapsulation efficiencies close to 100%. Floating microparticles consisting of polypropylene foam powder, model drug [chlorpheniramine maleate (CPM), diltiazem HCl, theophylline or verapamil HCl] and polymer [Eudragit RS or polymethyl methacrylate (PMMA)] were prepared by soaking the microporous foam carrier with an organic solution of drug and polymer and subsequent drying. The effects of various formulation and processing parameters on the resulting in vitro floating behaviour, internal and external particle morphology, drug loading, in vitro drug release and physical state of the incorporated drug were studied. Good in vitro floating behaviour was observed in most cases and a broad variety of drug release patterns could be achieved by varying the drug loading and type of polymer. Interestingly, PMMA-based microparticles showed incomplete drug release with verapamil HCl. This restriction could be overcome by forming the free base of the drug prior to microparticle preparation. In contrast to the salt, the free base acted as a plasticizer for PMMA, resulting in sufficiently high diffusion coefficients and, consequently, complete drug release. The low density microparticles were compressed into rapidly disintegrating tablets in order to provide an administrable oral dosage form.


Isolation and characterization of unsaturated fatty acid auxotrophs of Streptococcus pneumoniae and Streptococcus mutans.
Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.


Impaired Pavlovian conditioned inhibition in offenders with personality disorders.
Certain types of violent offending are often accompanied by evidence of personality disorders (PDs), a range of heterogeneous conditions characterized by disinhibited behaviours that are generally described as impulsive. The tasks previously used to show impulsivity deficits experimentally (in borderline personality disorder, BPD) have required participants to inhibit previously rewarded responses. To date, no research has examined the inhibition of responding based on Pavlovian stimulus-stimulus contingencies, formally "conditioned inhibition" (CI), in PDs. The present study used a computer-based task to measure excitatory and inhibitory learning within the same CI procedure in offenders recruited from the "personality disorder" and the "dangerous and severe personality disorder" units of a high-security psychiatric hospital. These offenders showed a striking and statistically significant change in the expression of inhibitory learning in a highly controlled procedure: The contextual information provided by conditioned inhibitors had virtually no effect on their prepotent associations. Moreover, this difference was not obviously attributable to nonspecific cognitive or motivational factors. Impaired CI would reduce the ability to learn to control associative triggers and so could provide an explanation of some types of offending behaviour.


A facile glovebox-free strategy to significantly accelerate the syntheses of well-defined polypeptides by N-carboxyanhydride (NCA) ring opening polymerizations.
A facile N2 flow-accelerated N-carboxyanhydride ring opening polymerization (NCA ROP) is demonstrated, herein, with rigorous kinetic studies to evaluate the methodology in detail. By using n-hexylamine as initiator and γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) as monomer, the NCA ROP via a normal amine mechanism (NAM) reached 90% conversion in 2 h under N2 flow at room temperature in a fume hood, much shorter than the time required for the same polymerization conducted in a glove box (14 h). The efficient removal of CO2 from the reaction by N2 flow drove the carbamic acid-amine equilibrium toward the formation of active nucleophilic amino termini and promoted polymerization. The detailed kinetic studies of the polymerization with different feed ratios and N2 flow rates were conducted, demonstrating the living feature of the NCA ROP and the tuning of the polymerization rate by simply changing the flow rate of N2. Maintenance of the reactivity of the amino ω-chain terminus and control during a subsequent polymerization were confirmed by performing chain extension reactions. The N2 flow method provides a new straightforward strategy to synthesize well-defined polypeptides with predictable molecular weights and narrow molecular weight distributions (PDI < 1.19).


UKPDS 19: heterogeneity in NIDDM: separate contributions of IRS-1 and beta 3-adrenergic-receptor mutations to insulin resistance and obesity respectively with no evidence for glycogen synthase gene mutations. UK Prospective Diabetes Study.
Insulin receptor substrate-1 (IRS-1), beta 3-adrenergic-receptor (beta 3-AR) and glycogen synthase (GS) genes are candidate genes for non-insulin-dependent diabetes mellitus (NIDDM), insulin resistance, dyslipidaemia and obesity. We studied white Caucasian subjects with NIDDM, 227 being randomly selected, 49 NIDDM within the top two percentiles of insulin resistance; 54 with dyslipidaemia in the top quintile of triglyceride/insulin and the bottom quintile of HDL, and 166 non-diabetic control subjects. We examined the association of the simple tandem repeat DNA polymorphisms (STRPs) near the IRS-1 and GS genes, and the prevalence of mutations at codons of IRS-1 513 and 972, beta 3-AR 64 and GS 464 using restriction fragment length polymorphism (RFLP). The STRP alleles in IRS-1 were significantly different between NIDDM and control subjects (p = 0.015). The IRS-1 972 mutation was significantly different between the four groups with increased prevalence in the insulin resistant and dyslipidaemia subjects (18 and 26% compared with 11% in control subjects; p < 0.0005). Those with or without IRS-1 mutations had similar clinical characteristics and impaired insulin sensitivity. beta 3-AR 64 mutation was not significantly different between the four groups but those with the mutation were more obese, with a test for linear association between number of alleles and degree of obesity in an analysis of variance showing a significant association (p = 0.029). The GS 464 mutation was not detected in any of the diabetic or control subjects and the population association study using GS STRP showed no difference in allelic frequencies between NIDDM patients and control subjects. A mutation in lipoprotein lipase at codon 291, associated in the general population with low HDL cholesterol, was not at increased prevalence in the NIDDM patients with dyslipidaemia. In conclusion, IRS-1 972 had an increased prevalence in subjects with insulin resistance, with or without dyslipidaemia. beta 3-AR 64 was associated with increased obesity but not with insulin resistance or dyslipidaemia. These separate contributions to different features of NIDDM are an example of the polygenic inheritance of this heterogeneous disorder.


Improved synthesis of 2'-deoxy-2'-[18F]fluoro-5-methyl-1-β-D-arabinofuranosyluracil ([18F]FMAU).
[18F]FMAU is an established PET probe used to monitor cellular proliferation. For clinical applications, a fully automated cGMP-compliant radiosynthesis would be preferred. However, the current synthesis of [18F]FMAU requires HBr activation of the sugar prior to the coupling with silylated uracil. This multiple step procedure makes the development of an automated protocol difficult and complicated. In this study, we report the use of Friedel-Crafts catalysts for an improved synthesis of [18F]FMAU, which also includes a significantly simplified one-pot reaction condition.


Influence of ingaalp laser (660nm) on the healing of skin wounds in diabetic rats.
To determine the influence of low-power laser (660 nm) on the collagen percentage and macrophages in skin wounds in diabetic rats. 30 male Wistar rats were used, distributed in two groups: laser treated diabetic (n= 15); untreated diabetic (n = 15). The diabetes was induced by intravenous injection of alloxan into the dorsal vein of the penis, at a rate of 0.1 ml of solution per 100 g of body weight. A wound was made on the back of all the animals. Groups 2 were treated with Aluminium Gallium Indium Phosphide - InGaAlP type diode laser (Photon Laser III DMC) with a continuous output power of 100 mW and wavelength (lambda) of 660 nm (4 J/cm(2)) for 24 s. five animal from each group was sacrificed on the 3rd, 7th and 14th days after wounding. Samples were taken, embedded in paraffin, stained with hematoxylin-eosin, Masson's trichrome, and immunohistochemical macrophage. morphometrically analyzed using the Image Pro Plus 4.5 software. The percentages of collagen fibers and macrophages were determined from the samples from the euthanasia animals. The data were treated statistically using analysis of variance (ANOVA) and the Post-hocTukey test. The significance level was set at 0.05 or 5%. The low-power laser (660 nm) was shown to be capable of influencing the collagen percentage in skin wounds by increasing the mean quantity of collagen fibers and macrophages.


Cell-specific expression of wild-type MeCP2 in mouse models of Rett syndrome yields insight about pathogenesis.
Rett syndrome (RTT), a leading cause of mental retardation with autistic features in females, is caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). RTT is characterized by a diverse set of neurological features that includes cognitive, motor, behavioral and autonomic disturbances. The diverse features suggest that specific neurons contribute to particular phenotypes and raise the question whether restoring MeCP2 function in a cell-specific manner will rescue some of the phenotypes seen in RTT. To address this, we generated transgenic mice expressing inducible MeCP2 under the control of the brain-specific promoters calcium/calmodulin-dependent protein kinase II (CamKII) or neuron-specific enolase (Eno2) and bred them onto mouse models lacking functional MeCP2. Expression of normal MeCP2 in either CamKII or Eno2 distribution was unable to prevent the appearance of most of the phenotypes of the RTT mouse models. These results suggest that most RTT phenotypes are caused either by disruption of complex neural networks involving neurons throughout the brain or by disruption of the function of specific neurons outside of the broad CamKII or Eno2 distribution.


Institutional development of freshwater fish stocking in Mexico.
By using freshwater fish stocking information from the Mexican government, this work described the current situation of the national stocking and its associated fishery policy. There is a lack of effective freshwater stocking programmes as a result of limited fisheries management, unharmonized fisheries regulations and institutional performance. The fry production has decreased from 140 to 20 million in the past 11 years.


Parthenogenetic chimaerism/mosaicism with a Silver-Russell syndrome-like phenotype.
We report a 34-year-old Japanese female with a Silver-Russell syndrome (SRS)-like phenotype and a mosaic Turner syndrome karyotype (45,X/46,XX). Molecular studies including methylation analysis of 17 differentially methylated regions (DMRs) on the autosomes and the XIST-DMR on the X chromosome and genome-wide microsatellite analysis for 96 autosomal loci and 30 X chromosomal loci revealed that the 46,XX cell lineage was accompanied by maternal uniparental isodisomy for all chromosomes (upid(AC)mat), whereas the 45,X cell lineage was associated with biparentally derived autosomes and a maternally derived X chromosome. The frequency of the 46,XX upid(AC)mat cells was calculated as 84% in leukocytes, 56% in salivary cells, and 18% in buccal epithelial cells. The results imply that a parthenogenetic activation took place around the time of fertilisation of a sperm missing a sex chromosome, resulting in the generation of the upid(AC)mat 46,XX cell lineage by endoreplication of one blastomere containing a female pronucleus and the 45,X cell lineage by union of male and female pronuclei. It is likely that the extent of overall (epi)genetic aberrations exceeded the threshold level for the development of SRS phenotype, but not for the occurrence of other imprinting disorders or recessive Mendelian disorders.


A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision.
Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.


[Activation of the brain and consciousness].
At least 3 levels of consciousness were identified within the stage B of sleep. Against the background of the increase in the alpha-rhythm amplitude, we observed an enhancement of voluntary and involuntary associations and increase in the flow of thoughts. The stage of "thought wandering" was accompanied by a considerable decrease in the alpha-rhythm regularity, a decrease in its frequency, and decrease in EEG coherence in all brain areas under study. At the stage of the "flat" EEG, the subjects experienced inhibition of mental processes ("empty heady"), their consciousness as if "falling down" to the lowest Ego level. The subjects were aware of the transition from one stage to another and could fix the stages. Thus, the results showed that subjective changes of levels of consciousness corresponded to different stages of transition from wakefulness to sleep which was objectively determined by the EEG.


Phase I dose-escalation studies of SNX-5422, an orally bioavailable heat shock protein 90 inhibitor, in patients with refractory solid tumours.
Orally administered SNX-5422, a novel, selective prodrug of the Heat shock protein 90 (Hsp90) inhibitor SNX-2112, was investigated in two sequential phase I studies to determine the safety, maximum tolerated doses (MTDs) and pharmacokinetic profile of SNX-5422. Using a dose-escalation design, 3-6 adults with advanced solid tumours received SNX-5422 every-other-day (QOD) or once-daily (QD) 3weeks on/1week off or QD continuously, with disease assessments every 8 weeks. Single-dose and steady-state pharmacokinetic parameters of SNX-2112 were determined. In total, 56 patients were enrolled: QOD 3 weeks on/1 week off, n=36; QD 3weeks on/1 week off, n=17; QD continuous, n=3. Doses ranged from 4 to 133mg/m(2) QOD and 50 to 89 mg/m(2) QD. The MTDs were defined as 100mg/m(2) QOD and 67 mg/m(2) QD, respectively, with diarrhoea being dose-limiting on both 3 weeks on/1 week off schedules. Overall, treatment-related adverse events were mainly low grade, including diarrhoea (64%), nausea (39%), fatigue (28%), and vomiting (28%). Reversible grade 1-3 nyctalopia (night blindness) was reported by four patients (dose: 50-89mg/m(2) QD; 100mg/m(2) QOD). Exposure was generally linear, though greater than dose-proportional. Of 32 evaluable patients on QOD dosing, there was one durable complete response (prostate cancer), one confirmed (HER2+breast cancer) and one unconfirmed partial response (adrenal gland cancer). Three patients (QOD schedule) had stable disease for ⩾ 6 months. The dose and schedule recommended for further study with SNX-5422 is 100mg/m(2) QOD 3 weeks on/1 week off based on improved tolerability and preliminary evidence of clinical activity.


Computer-assisted image analysis of bronchioloalveolar carcinoma.
In clinical trials, response rate is an important endpoint for assessing the efficacy of an anticancer drug. The Response Evaluation Criteria for Solid Tumors (RECIST) has been widely used as a standard method to assess response. The RECIST requires only 1-dimensional measurement of tumor size. However, bronchioloalveolar carcinoma (BAC), which commonly presents as infiltrative or micronodular lesions, is not always readily assessable by RECIST. During the past 2 years, we have been developing computer-based programs to more accurately measure tumor size on chest computed tomography (CT) scans. In a first-generation computer-assisted image analysis (CAIA) system, we were able to capture and quantify lesions on CT scans by linking the software programs of eFilm, HyperSnap, and Scion. We have applied this CAIA approach to measuring BAC response to gefitinib in the Southwest Oncology Group (S0126) trial. However, this first-generation CAIA system involves multiple manual steps and is therefore labor intensive. We are now developing a fully automated CAIA program based on a versatile software platform, ImageJ, created at the National Institutes of Health. Taking theoretical and physical considerations into account, Java plug-in programs for ImageJ are created to automatically analyze CT scans in the Digital Imaging and Communications in Medicine format. We have demonstrated the feasibility of an ImageJ-based automated CAIA program for measuring BAC bidimensionally on CT scans. This automated CAIA system will be applied in a prospective clinical trial of the GVAX vaccine in patients with BAC.


Microfluidic thrombosis under multiple shear rates and antiplatelet therapy doses.
The mainstay of treatment for thrombosis, the formation of occlusive platelet aggregates that often lead to heart attack and stroke, is antiplatelet therapy. Antiplatelet therapy dosing and resistance are poorly understood, leading to potential incorrect and ineffective dosing. Shear rate is also suspected to play a major role in thrombosis, but instrumentation to measure its influence has been limited by flow conditions, agonist use, and non-systematic and/or non-quantitative studies. In this work we measured occlusion times and thrombus detachment for a range of initial shear rates (500, 1500, 4000, and 10000 s(-1)) and therapy concentrations (0-2.4 µM for eptifibatide, 0-2 mM for acetyl-salicylic acid (ASA), 3.5-40 Units/L for heparin) using a microfluidic device. We also measured complete blood counts (CBC) and platelet activity using whole blood impedance aggregometry. Effects of shear rate and dose were analyzed using general linear models, logistic regressions, and Cox proportional hazards models. Shear rates have significant effects on thrombosis/dose-response curves for all tested therapies. ASA has little effect on high shear occlusion times, even at very high doses (up to 20 times the recommended dose). Under ASA therapy, thrombi formed at high shear rates were 4 times more prone to detachment compared to those formed under control conditions. Eptifibatide reduced occlusion when controlling for shear rate and its efficacy increased with dose concentration. In contrast, the hazard of occlusion from ASA was several orders of magnitude higher than that of eptifibatide. Our results show similar dose efficacy to our low shear measurements using whole blood aggregometry. This quantitative and statistically validated study of the effects of a wide range of shear rate and antiplatelet therapy doses on occlusive thrombosis contributes to more accurate understanding of thrombosis and to models for optimizing patient treatment.


Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulence.
Yersinia ruckeri is a Gram-negative pathogen that causes enteric redmouth disease in salmonids. A gene from Y. ruckeri encoding an extracellular protease termed yrp1 (Yersinia ruckeri protease 1) was cloned from a Sau3AI library constructed in pUC19 and analysed in gelatin-supplemented medium. The nucleotide sequence of the yrp1 gene indicated an ORF encoding a protein of 477 aa. On the basis of the high degree of homology in the amino acid sequence as well as its conservative motifs, this protein was included within the serralysin metalloendopeptidase subfamily (EC 184.108.40.206). The yrp1 N-terminal sequence showed a 14 aa propeptide followed by a 10 aa sequence identical to the one deduced previously from the 47 kDa purified protease. Additional results demonstrated that the yrp1 gene encodes the 47 kDa protein. In contrast to other Yersinia species, the yrp1 protease is secreted by a type I Gram-negative bacterial ABC exporter protein secretion system composed of three genes termed yrpD, yrpE and yrpF, and a protease inhibitor inh. The development of genetic methods for this species has allowed the exploration of the organization and the putative role of the Yrp1 genetic locus. Thus, site-directed insertion mutations into the yrp1 and the yrpE genes were constructed by the integration of the mobilizable suicide vector pIVET8 containing internal portions of both coding sequences. Complementation studies of those mutants with different loci indicated that they are organized as a single operon. The mutant strains lacked protease activity as well as the Yrp1 protein and, although physiologically similar to the parental strain when growing on nutrient broth medium, they were attenuated in virulence when bacteria were injected intraperitoneally into rainbow trout (Oncorhynchus mykiss). This is the first report of defined mutations in Y. ruckeri to show the implication of a factor such as an extracellular protease in pathogenesis.


Evidence that glutathione S-transferases B1B1 and B2B2 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the B1 and B2 subunits and other Alpha class glutathione S-transferases.
The Alpha class glutathione S-transferases (GSTs) in human liver are composed of polypeptides of Mr 25,900. These enzymes are dimeric, and two immunochemically distinct subunits, B1 and B2, have been described that combine to form GSTs B1B1, B1B2 and B2B2 [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Gradient affinity elution from GSH-Sepharose has been used to resolve the three Alpha class GSTs, and this method has been applied to demonstrate marked inter-individual differences in the hepatic content of GSTs B1B1, B1B2 and B2B2. The B1 and B2 subunits can be resolved by reverse-phase h.p.l.c., and their elution positions suggest that they are equivalent to the alpha chi and alpha y h.p.l.c. peaks described by Ketterer and his colleagues [Ostlund Farrants, Meyer, Coles, Southan, Aitken, Johnson & Ketterer (1987) Biochem. J. 245, 423-428]. The B1 and B2 subunits have now been cleaved with CNBr and the fragments subjected to automated amino acid sequence analysis. The sequence data show that B1 and B2 subunits do not arise from post-translational modification, as had been previously believed for the hepatic Alpha class GSTs, but are instead the products of separate genes; B1 and B2 subunits were found to contain different amino acid residues at positions 88, 110, 111, 112, 116, 124 and 127. The relationship between the B1 and B2 subunits and the cloned GTH1 and GTH2 cDNA sequences [Rhoads, Zarlengo & Tu (1987) Biochem. Biophys. Res. Commun. 145, 474-481] is discussed.


[Alzheimer's disease and methylenetetrahydrofolate reductase gene polymorphisms: a potential nutrigenomic approach for Mexico].
The establishment of medical genomics in Mexico offers the possibility to study in a more comprehensive manner the etiological factors of different diseases, providing a global view of the interaction between the genome and the environment. Nutrition is recognized as a significant determinant in several diseases, yet its interaction with polymorphisms, and in general with the genome, has not been properly addressed Mexico has a high prevalence of polymorphisms of the methylenetetrahydrofolate reductase gene, and in both clinical and basic studies this has been associated with an increased susceptibility of developing Alzheimer's disease. We propose a potential nutrigenomic approach for the study of Alzheimer disease in Mexico.


Aire-dependent alterations in medullary thymic epithelium indicate a role for Aire in thymic epithelial differentiation.
The prevalent view of thymic epithelial differentiation and Aire activity holds that Aire functions in terminally differentiated medullary thymic epithelial cells (MTECs) to derepress the expression of structural tissue-restricted Ags, including pancreatic endocrine hormones. An alternative view of these processes has proposed that Aire functions to regulate the differentiation of immature thymic epithelial cells, thereby affecting tissue-restricted Ag expression and negative selection. In this study, we demonstrate that Aire impacts several aspects of murine MTECs and provide support for this second model. Expression of transcription factors associated with developmental plasticity of progenitor cells, Nanog, Oct4, and Sox2, by MTECs was Aire dependent. Similarly, the transcription factors that regulate pancreatic development and the expression of pancreatic hormones are also expressed by wild-type MTECs in an Aire-dependent manner. The altered transcriptional profiles in Aire-deficient MTECs were accompanied by changes in the organization and composition of the medullary epithelial compartment, including a reduction in the medullary compartment defined by keratin (K) 14 expression, altered patterns of K5 and K8 expression, and more prominent epithelial cysts. These findings implicate Aire in the regulation of MTEC differentiation and the organization of the medullary thymic compartment and are compatible with a role for Aire in thymic epithelium differentiation.


Myoferlin regulation by NFAT in muscle injury, regeneration and repair.
Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.


The activation of presynaptic choline uptake by acetylcholine release.
1. Acetylcholine release evoked from nerve terminals in vivo or in vitro results in a subsequent activation of choline uptake. 2. The activation of choline transport is not due to any obligatory coupling with acetylcholine synthesis because incoming choline is diluted with endogenous choline. 3. The choline carrier demonstrates trans-activation, i.e. influx stimulates efflux and vice versa. Acetylcholine is a weak competitor for the choline binding site and is hardly transported. 4. The binding of acetylcholine without concomitant transport has the effect of immobilizing the carrier on the side of the high acetylcholine concentration inside the terminal so that it is not available for the transport of choline from the outside. When intraterminal acetylcholine concentration is reduced by the release process the immobilization of the carrier is relieved thus enabling it to return to the outside of the membrane and activate choline uptake. 5. The choline carrier has been solubilized and incorporated into an artificial membrane system (liposomes). In liposomes the carrier demonstrates saturability, inhibition by hemicholinium-3 and trans-activation. The choline transported is not acetylated nor does S-acetyl-CoA increase the rate. Influx is stimulated by Na+ on the outside and K+ on the inside.


[Identification and characterization of marker chromosome in Turner syndrome].
To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome, and study the phenotypic effects resulting from the abnormal karyotype. Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome, and 6 marker chromosomes were ring chromosomes. Their karyotypes were showed as mos. 45, X/46, X, + mar or mos. 45, X/46, X, + r. Fluorescence in situ hybridization (FISH) technique with X/Y centromere probes was performed to determine the origin of the marker chromosome. Reverse chromosome painting technique was used to identify the breakpoints of two largest markers. Phenotype effects with different chromosome breakpoints were compared. All the 11 marker chromosomes were ring X chromosomes. The breakpoints of the r(X) were involved in Xp22, Xq22, Xq24 and Xq26, etc. The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X. Each r(X) in our patients was mosaic, indicating it was originated from mitosis error during early embryo development. To analyze the origin of the marker chromosome and the breakpoint of r(X) will provide guidance for the therapy and prognosis of the Turner syndrome patient.


The migration behavior of human glioblastoma cells is influenced by the redox-sensitive human macrophage capping protein CAPG.
The macrophage capping protein CAPG belongs to the gelsolin superfamily which modulates actin dynamics by capping the growing end of actin filaments in a Ca2+- and PIP2-dependent manner resulting in polymerization inhibition of actin filaments. In the last years, additional functions for CAPG in transcription regulation were described and higher CAPG amounts have been linked to increased invasiveness and migration behavior in different human tumor entities like e.g. glioblastoma. Nevertheless, there is a lack of knowledge how additional functions of CAPG are regulated. As CAPG contains several cysteine residues which may be accessible to oxidation we were especially interested to investigate how alterations in the cysteine oxidation state may influence the function, localization, and regulation of CAPG. In the present study, we provide strong evidence that CAPG is a redox-sensitive protein and identified two cysteines: C282 and C290 as reversibly oxidized in glioblastoma cell lines. Whereas no evidence could be found that the canonical actin capping function of CAPG is redox-regulated, our results point to a novel role of the identified cysteines in the regulation of cell migration. Along with this, we found a localization shift out of the nucleus of CAPG and RAVER1, a potential interaction partner identified in our study which might explain the observed altered cell migration properties. The newly identified redox sensitive cysteines of CAPG could perspectively be considered as new targets for controlling tumor invasive properties.


Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes.
A stable QTL that may be used in marker-assisted selection in wheat breeding programs was detected for yield, yield components and drought tolerance-related traits in spring wheat association mapping panel. Genome-wide association mapping has become a widespread method of quantitative trait locus (QTL) identification for many crop plants including wheat (Triticum aestivum L.). Its benefit over traditional bi-parental mapping approaches depends on the extent of linkage disequilibrium in the mapping population. The objectives of this study were to determine linkage disequilibrium decay rate and population structure in a spring wheat association mapping panel (n = 285-294) and to identify markers associated with yield and yield components, morphological, phenological, and drought tolerance-related traits. The study was conducted under fully irrigated and rain-fed conditions at Greeley, CO, USA and Melkassa, Ethiopia in 2010 and 2011 (five total environments). Genotypic data were generated using diversity array technology markers. Linkage disequilibrium decay rate extended over a longer genetic distance for the D genome (6.8 cM) than for the A and B genomes (1.7 and 2.0 cM, respectively). Seven subpopulations were identified with population structure analysis. A stable QTL was detected for grain yield on chromosome 2DS both under irrigated and rain-fed conditions. A multi-trait region significant for yield and yield components was found on chromosome 5B. Grain yield QTL on chromosome 1BS co-localized with harvest index QTL. Vegetation indices shared QTL with harvest index on chromosome 1AL and 5A. After validation in relevant genetic backgrounds and environments, QTL detected in this study for yield, yield components and drought tolerance-related traits may be used in marker-assisted selection in wheat breeding programs.


Decrypting the role of Cripto in tumorigenesis.
The cell-surface associated molecule Cripto is overexpressed in a wide range of epithelial cancers, yet little is known about potential mechanisms by which Cripto expression might enhance tumorigenesis. A new study reveals that binding of Cripto to the TGF-beta ligand Activin B can block Activin B-mediated suppression of cell proliferation. Furthermore, this study also demonstrates that antibody blockade of Cripto function may prove useful in the inhibition of tumorigenesis.


Clinical Characteristics of Patients Experiencing Pathologic Complete Response Following Neoadjuvant Therapy for Borderline Resectable/Locally Advanced Pancreatic Adenocarcinoma.
The purpose of this study is to describe clinical characteristics and outcomes of patients with borderline resectable pancreatic cancer (BRPC) or locally advanced pancreatic cancer (LAPC) who achieved pathologic complete response (pCR) following neoadjuvant therapy. A single institution clinical database for patients with pancreatic ductal adenocarcinoma was queried. Between 2008 and 2014 patients were identified with BRPC and LAPC, who underwent surgical resection after receiving neoadjuvant treatment. Clinical and pathologic features of the patients who achieved pCR were acquired retrospectively. Six patients were identified to have pCR on pathology of the postoperative specimen. On the basis of pretreatment clinical staging, 2 patients were considered to have BRPC and 4 LAPC. Four patients received gemcitabine-based chemotherapy and 2 patients received FOLFIRINOX (5-fluorouracil, oxaliplatin, irinotecan, and leucovorin). Five of 6 patients received radiation therapy before operative resection. Operative procedures included distal pancreatectomy (n=3) and pancreatoduodenectomy (n=3). Pancreatic intraepithelial neoplasia 1 to 2 was present in 3 cases, and pancreatic intraepithelial neoplasia 3 in 1 case. During a median follow-up of 21.3 months, 2 patients died, with a median survival of 11.0 months (range, 10.4 to 11.6 mo). Four patients are alive and continue to follow-up with median survival of 28.7 months (range, 20.1 to 42.4 mo). Multimodality neoadjuvant therapy may lead to complete pathologic response in a small number of patients with borderline resectable/locally advanced pancreatic adenocarcinoma. pCR to neoadjuvant therapy does not lead to cure in most cases, and the majority of patients appear to relapse locally or systemically.


Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes.
Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal-nuclear-chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.


Clinical outcomes using standard phacoemulsification and femtosecond laser-assisted surgery with toric intraocular lenses.
To compare the 1-month and 1-year results of toric intraocular lens (IOL) implantation with standard (manual) phacoemulsification vs femtosecond laser-assisted surgery. Refractive data, visual acuity data, and ocular aberration measured with a wavefront aberrometer were collected for two groups of patients from one site. The first group had standard phacoemulsification, while the second group had femtosecond laser-assisted surgery, and both groups were implanted with toric IOLs, either monofocal or multifocal. Differences in visual acuity, refractive outcomes, and higher order aberrations - total, corneal, and internal - were evaluated at 1 month and 1 year postoperatively. Toric IOLs were implanted in 62 eyes using standard phacoemulsification and 53 eyes using femtosecond laser-assisted surgery. Uncorrected visual acuity and best-spectacle-corrected visual acuity at 1 month and 1 year were not statistically significantly different between the groups (P>0.05) nor was the mean cylinder or mean spherical equivalent refraction (P>0.12). Total ocular higher order aberrations were significantly different between the groups (P<0.05), but absolute differences appeared to be the same. Internal vertical coma was significantly lower in the femto group at 1 year (P=0.03). Differences in aberrations did not correlate with corrected or uncorrected visual acuity. Patients who underwent uncomplicated lens surgery with toric IOLs in both the groups had comparable refractive outcomes in terms of visual acuity and residual refraction at 1 year. The femto group had significantly lower internal vertical coma at 1 year.


Cisplatin plus paclitaxel and bevacizumab versus carboplatin plus paclitaxel and bevacizumab for the first-line treatment of metastatic or recurrent cervical cancer.
Cisplatin-paclitaxel and bevacizumab is a frequently used treatment regimen for metastatic or recurrent cervical cancer, and carboplatin-paclitaxel and bevacizumab are also among the recommended regimens. In this study we aimed to evaluate the efficacy of these two regimens for the treatment of metastatic or recurrent cervical cancer. Patients with metastatic or recurrent cervical cancer treated with cisplatin-paclitaxel and bevacizumab or carboplatin-paclitaxel and bevacizumab were retrospectively evaluated in this study. The clinical and demographic characteristics of patients in each group were evaluated. Median overall survival, progression-free survival, and response rates between the two groups were compared. A total of 250 patients were included. Overall, the numbers of patients with recurrent disease and metastatic disease were 159 and 91, respectively. The most common histologic subtype was squamous cell carcinoma (83.2%). The median duration of follow-up was 13.6 (range 0.5-86) months. The median progression-free survival was 10.5 (95% CI 9.0 to 11.8) months in the cisplatin-paclitaxel and bevacizumab group (group 1), and 10.8 (95% CI 8.6 to 13.0) months in the carboplatin-paclitaxel and bevacizumab group (group 2) (HR 1.20; 95% CI 0.88 to 1.63; p=0.25). The median overall survival was 19.1 (95% CI 13.0 to 25.1) months in group 1 and 18.3 (95% CI 15.3 to 21.3) months in group 2 (HR 1.28; 95% CI 0.91 to 1.80; p=0.15). There is no survival difference between cisplatin or carboplatin combined with paclitaxel and bevacizumab in metastatic or recurrent cervical cancer.


[Unknown hepatoid glands of some species of cats and deer].
Our histological description of the fourth type of mammalian skin glands--hepatiod glands--allowed us to revise the obtained and published data mistaking the alveolar glands of the anal sacs in cats and the limb glands in deers as sebaceous. Large clusters of hepatiod glands were discovered in the anal sacs of the cat, lynx, and tiger, interdigital gland of elk, and the tarsal gland of reindeer. These glands secrete considerable amounts of protein to the clearance of the intercellular canaliculi and contain hydrophobic lipids. The available data substantiate revision of the data on the structure of many skin glandular organs with atypical sebaceous glands.


Inspiratory muscle training in children and adolescents living with neuromuscular diseases: A pre-experimental study.
Children with neuromuscular diseases (NMD) are at risk of morbidity and mortality because of progressive respiratory muscle weakness and ineffective cough. Inspiratory muscle training (IMT) aims to preserve or improve respiratory muscle strength, thereby reducing morbidity and improving health-related quality of life (HRQoL). To describe the safety and feasibility of a 6-week IMT programme using an electronic threshold device (Powerbreathe®). Any adverse events and changes in functional ability, spirometry, peak expiratory cough flow (PECF), inspiratory muscle strength and HRQoL (Pediatric Quality of Life [PedsQL]) were recorded. A convenience sample of eight participants (n = 4 boys; median [interquartile range {IQR}] age: 12.21 [9.63-16.05] years) with various NMD were included in a pre-experimental, observational pre-test post-test feasibility study. Training consisted of 30 breaths, twice daily, 5 days a week, for 6 weeks. There were significant pre- to post-intervention improvements in upper limb function and coordination (p = 0.03) and inspiratory muscle strength: maximum inspiratory mouth pressure (Pimax) (p = 0.01); strength-index (p = 0.02); peak inspiratory flow (PIF) (p = 0.02), with no evidence of change in spirometry, PECF or HRQoL. No adverse events occurred and participant satisfaction and adherence levels were high. Inspiratory muscle training (at an intensity of 30% Pimax) appears safe, feasible and acceptable, in a small sample of children and adolescents with NMD and was associated with improved inspiratory muscle strength, PIF and upper limb function and coordination. Larger, longer-term randomised controlled trials are warranted to confirm the safety and efficacy of IMT as an adjunct respiratory management strategy in children with NMD.


Whole-genome screening in ankylosing spondylitis: evidence of non-MHC genetic-susceptibility loci.
Ankylosing spondylitis (AS) is a common inflammatory arthritis predominantly affecting the axial skeleton. Susceptibility to the disease is thought to be oligogenic. To identify the genes involved, we have performed a genomewide scan in 185 families containing 255 affected sibling pairs. Two-point and multipoint nonparametric linkage analysis was performed. Regions were identified showing "suggestive" or stronger linkage with the disease on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q. The MHC locus was identified as encoding the greatest component of susceptibility, with an overall LOD score of 15.6. The strongest non-MHC linkage lies on chromosome 16q (overall LOD score 4.7). These results strongly support the presence of non-MHC genetic-susceptibility factors in AS and point to their likely locations.


Discovery of till deposition at the grounding line of Whillans Ice Stream.
We report on the discovery of a grounding-line sedimentary wedge ("till delta") deposited by Whillans Ice Stream, West Antarctica. Our observation is that grounding-line deposition serves to thicken the ice and stabilize the position of the grounding line. The ice thickness at the grounding line is greater than that of floating ice in hydrostatic equilibrium. Thus, the grounding line will tend to remain in the same location despite changes in sea level (until sea level rises enough to overcome the excess thickness that is due to the wedge). Further, our observation demonstrates the occurrence of rapid subglacial erosion, sediment transport by distributed subglacial till deformation, and grounding-line sedimentation, which have important implications for ice dynamics, numerical modeling of ice flow, and interpretation of the sedimentation record.


Outcomes following ossicular chain reconstruction with composite prostheses: hydroxyapatite-polyethylene vs. hydroxyapatite-titanium.
We conducted a retrospective study to compare the results of ossicular chain reconstruction (OCR) with two types of composite prosthesis: a hydroxyapatite-polyethylene (HAPEX) implant and a hydroxyapatite-titanium (HATi) prosthesis. We reviewed the records of 222 patients-104 males and 118 females, aged 8 to 79 years (mean: 39.7)-who had undergone OCR for ossicular chain dysfunction and who met our eligibility criteria. In addition to demographic data and the type of prosthesis, we compiled information on pre- and postoperative audiometric findings, the underlying diagnosis, the timing of surgery (primary, planned, or revision), the type of surgery (tympanoplasty alone, tympanoplasty with antrotomy, intact-canal-wall tympanomastoidectomy, or canal-wall-down tympanomastoidectomy), the extent of reconstruction (partial or total), the use of the malleus, the use of a tragal cartilage graft, and evidence of extrusion. Of the 222 patients, 46 had undergone insertion of either a partial (n = 36) or total (n = 10) ossicular replacement prosthesis (PORP and TORP, respectively) made with HAPEX, and 176 had received a PORP (n = 101) or TORP (n = 75) made with HATi. Postoperatively, the mean air-bone gap (ABG) was 14.0 dB in the HAPEX group and 14.7 dB in the HATi group, which was not a significant difference (p = 0.61). Postoperative success (ABG ≤20 dB) with PORP was obtained in 30 of the 36 patients in the HAPEX group (83.3%) and in 87 of the 101 patients in the HATi group (86.1%), while success with TORP was achieved in 7 of 10 HAPEX patients (70.0%) and 56 of 75 HATi patients (74.7%); there was no significant difference in either PORP or TORP success rates between the HAPEX and HATi groups (p = 0.32). A significantly better hearing result was obtained when the malleus was used in reconstruction (p = 0.035), but the use of tragal cartilage led to a significantly worse outcome (p = 0.026). Revision surgery was associated with a significantly worse postoperative result (p = 0.034). Prosthesis extrusion was observed in 9.0% of all cases. The two types of composite assessed in this study yielded similar results in terms of functional hearing and stability, but the HATi prosthesis had some significant advantages. For example, it was associated with more cases in which the ABG closed to less than 10 dB. In addition, because of its thinner stem and lower profile, it can be used in situations that are not possible with the HAPEX implant.


Effects of tetrahydroprotoberberines on dopamine D2 receptors in ventral tegmental area of rat.
To compare the actions of tetrahydroprotoberberines (THPB) on dopamine (DA) D2 receptors in the ventral tegmental area (VTA) of rat. Extracellular single unit recording technique was used in i.v. gallamine-paralyzed rats. Eleven THPB analogs tested completely attenuated the apomorphine (Apo, 20 micrograms.kg-1)-induced inhibition on VTA DA cell firing activity. The OH group on C2 at THPB was linked with the reversal of Apo-induced inhibition. Their reversal potencies (ED50, microgram.kg-1) for D2 receptors were: THPB-143 (5.6) > SPD (8.5) > Iso (17.0) > THP (33) > THB (48) > THPB-18 (66) > THPB-1 (179) > THPB-19 (408) > THPB-126 (510) > THPB-104 (1019) > THPB-10 (4815). Among these 11 THPB, the 2-hydroxyl-THPB (THPB-143) showed the strongest antagonistic action on D2 receptors.


The Alabama Eye Bank: 25 years of giving sight.
Modern Eye Banking had its start in the mid-1940's. However, it was not until the 1960's that eye banking grew to become a movement or even a necessary part of the health care delivery system in the state of Alabama. The Alabama Eye Bank has grown rapidly to become one of the largest corneal processing networks in the United States. Corneal transplant surgery remains highly successful in restoring sight. The contraindications for donor tissue should be clarified to all physicians and other health care professionals. The public should be make aware of the vision care available to them in the state of Alabama. The general public should be aware of how to become a pre-pledged eye donor, of research, teaching and transplantable tissue and also how they can be assured that their wishes are carried out at the time of their demise. Even with scheduled transplant surgery for all Alabamians, the need for the general public and medical professionals to be oriented and educated towards multi-organ and tissue donations is important.


[Significance of various antiphysiological and nutritional factors on the biological evaluation of various cultures of the common bean (Phaseolus sp.)].
Raw and cooked samples of beans supplemented with methionine were used to prepare 35 rat diets containing different amounts of methionine, tannins, and trypsin inhibitors. The effect of these diets on weight gain, mortality and protein utilization was measured. The weight gain/protein consumption ratio increased while mortality decreased as the proportion of raw beans decreased in the diet. The effects of diet, beans and their interaction were significant. Supplementation with methionine had no effect in diets with high proportions of raw beans, but it had a significant effect on the biological response to diets with high proportions of cooked beans. The high and early mortality that occurred with diets with a high content of raw beans did not permit the adequate determination of apparent digestibility. In diets with a high concentration of cooked beans, digestibility was higher for white than for black or red beans. Since methionine supplementation had no effect on digestibility, it is suggested that heat-induced changes in the protein could be of great importance in the improvement of bean protein. At the same time, heat destroys the antiphysiological factors, since no definite detrimental effect was observed in the cooked samples due to these factors. It is concluded that the low nutritive value of cooked beans is due to a deficiency of sulfur-containing amino acids, and that the structure of the protein is a factor of the utmost importance in determining its digestibility.


Impact of placing a conduit from the right ventricle to the pulmonary arteries as the first stage of further palliation in the Norwood sequence for hypoplasia of the left heart.
We describe the experience from a single institution with the Norwood sequence of palliation for hypoplasia of the left heart, emphasizing complications related to placement of a conduit from the right ventricle to the pulmonary arteries and their management. Between November, 2002 and January, 2006, we palliated 32 patients with hypoplastic left heart syndrome or its variants by placing a conduit from the right ventricle to the pulmonary arteries. We reviewed retrospectively the charts and angiograms from these patients. Hospital survival after construction of the conduit was 90.6%. There were 3 interstage deaths, of which 2 were likely due severe obstruction of the conduit. Stents were implanted into the proximal or medial portions of the conduits of 3 patients. Early revision of the distal anastomosis, and shortening the conduit, was performed early postoperatively in 2 patients. So far, 24 out 26 survivors of the first stage underwent a bi-directional cavopulmonary anastomosis after a mean interval of 4.3 plus or minus 1.4 months. Of these, 3 required a semi-urgent second stage of palliation because of worsening cyanosis, with one patient dying after the second stage. Completion of the Fontan circulation by insertion of an extracardiac conduit was performed in 8 patients at the mean age of 19.8 plus or minus 2.2 months. We were able to achieve biventricular repair in 1 patient, with aortic atresia, hypoplastic arch and ventricular septal defect, 4.3 months after the initial palliative procedure. Overall survival of the whole cohort of 32 patients was 78.9%, plus or minus 7.8%, at 5 months, and 74.3%, plus or minus 8.6%, up to 25 months. The introduction of the conduit placed from the right ventricle to the pulmonary arteries has led to an improved outcome in the complex entity of hypoplastic left heart syndrome and its variants. Stenosis of the conduit, nonetheless, may account for significant interstage morbidity, and often requires intervention or early installation of the second stage of palliation.


SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.


Expanded cardiovascular phenotype of Myhre syndrome includes tetralogy of Fallot suggesting a role for SMAD4 in human neural crest defects.
Tetralogy of Fallot (ToF) can be associated with a wide range of extracardiac anomalies, with an underlying etiology identified in approximately 10% of cases. Individuals affected with Myhre syndrome due to recurrent SMAD4 mutations frequently have cardiovascular anomalies, including congenital heart defects. In addition to two patients in the literature with ToF, we describe five additional individuals with Myhre syndrome and classic ToF, ToF with pulmonary atresia and multiple aorto-pulmonary collaterals, and ToF with absent pulmonary valve. Aorta hypoplasia was documented in one patient and suspected in another two. In half of these individuals, postoperative cardiac dysfunction was thought to be more severe than classic postoperative ToF repair. There may be an increase in right ventricular pressure, and right ventricular dysfunction due to free pulmonic regurgitation. Noncardiac developmental abnormalities in our series and the literature, including corectopia, heterochromia iridis, and congenital miosis suggest an underlying defect of neural crest cell migration in Myhre syndrome. We advise clinicians that Myhre syndrome should be considered in the genetic evaluation of a child with ToF, short stature, unusual facial features, and developmental delay, as these children may be at risk for increased postoperative morbidity. Additional research is needed to investigate the hypothesis that postoperative hemodynamics in these patients may be consistent with restrictive myocardial physiology.


Limb Amputations in Cancer: Modern Perspectives, Outcomes, and Alternatives.
This review summarizes current findings regarding limb amputation within the context of cancer, especially in osteosarcomas and other bony malignancies. We seek to answer the question of how amputation is utilized in the contemporary management of cancer as well as explore current advances in limb-sparing techniques. The latest research on amputation has been sparse given its extensive history and application. However, new research has shown that rotationplasty, osseointegration, targeted muscle reinnervation (TMR), and regenerative peripheral nerve interfaces (RPNI) can provide patients with better functional outcomes than traditional amputation. While limb-sparing surgeries are the mainstay for managing musculoskeletal malignancies, limb amputation is useful as a palliative technique or as a primary treatment modality for more complex cancers. Currently, rotationplasty and osseointegration have been valuable limb-sparing techniques with osseointegration continuing to develop in recent years. TMR and RPNI have also been of interest in the modern management of patients requiring full or partial amputations, allowing for better control over myoelectric prostheses.


Do infants really expect agents to act efficiently? A critical test of the rationality principle.
Recent experiments have suggested that infants' expectations about the actions of agents are guided by a principle of rationality: In particular, infants expect agents to pursue their goals efficiently, expending as little effort as possible. However, these experiments have all presented infants with infrequent or odd actions, which leaves the results open to alternative interpretations and makes it difficult to determine whether infants possess a general expectation of efficiency. We devised a critical test of the rationality principle that did not involve infrequent or odd actions. In two experiments, 16-month-olds watched events in which an agent faced two identical goal objects; although both objects could be reached by typical, everyday actions, one object was physically (Experiment 1) or mentally (Experiment 2) more accessible than the other. In both experiments, infants expected the agent to select the more-accessible object. These results provide new evidence that infants possess a general and robust expectation of efficiency.


Effective elimination of acute myeloid leukemic cells by recombinant bispecific antibody derivatives directed against CD33 and CD16.
Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.


Unravelling the complexity of T cell abnormalities in common variable immunodeficiency.
We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.


Episodes of psychiatric care and medical utilization.
This study examines the reduction in medical care utilization after mental health treatment, also known as the offset effect. With data from a computerized ambulatory care information system, an episode-of-care method is used to characterize the mental health care received by patients in a prepaid group practice. The characteristics of the psychiatric episodes, such as duration, type of therapy, and number of visits, are examined in relation to the degree of offset effects among psychiatric patients. The results indicate that offset effects are most pronounced for a variety of characteristics related to the psychiatric episode of care including brief, high-intensity therapy, treatment for transient rather than chronic mental illness, and individual rather than group therapies. Finally, this study replicates two major findings in the offset literature: the characteristic peaking of medical use before mental health care and the short-term nature of the offset effect.


The anti-proliferative inhibition of ellipticine in human breast mda-mb-231 cancer cells is through cell cycle arrest and apoptosis induction.
Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here we report the mechanism of apoptosis induction and cell cycle arrest by ellipticine in human breast MDA-MB-231 cancer cells. Ellipticine treatment arrested MDA-MB-231 cells at the G2/M phase after 6 h of treatment. This effect was strongly associated with a concomitant decrease in the level of cyclin B1, Cdc25 and Cdc2, and increase in phospho-Cdc2 (Tyr15). In addition, ellipticine also induced apoptosis in MDA-MB-231 cells, as determined by using both DNA fragmentation and Annexin-V staining assay. Ellipticine increased the expression of Bax, but decreased the level of Bcl-2, Bcl-XL and X-linked inhibitor of apoptosis protein (XIAP), and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c, and activation of caspase-9 and -3). In addition, pre-treatment of cells with caspase-9 inhibitor inhibited ellipticine-induced cell proliferation and apoptosis, indicating that caspase-9 activation was involved in MDA-MB-231 cell apoptosis induced by ellipticine. Taken together, our study suggests that the inhibition of cell cycle progression signaling and initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of ellipticine in MDA-MB-231 cells.


Feasibility of limb salvage and survival in soft tissue sarcomas.
One hundred nine consecutive patients with soft tissue sarcomas were treated in the period 1977 through 1983. Of 85 patients with extremity sarcomas, only 3 patients (4%) were managed with amputation, whereas in the previous decade, 40% of such patients were treated with amputation in our institute. The current 5-year survival rate is 63%; in the previous decade it was 45%. In the current series, for extremity locations, patients with minimum surgical margins of 2 cm or greater and no further local therapy had a 5-year local recurrence rate of 17%, whereas those with minimum surgical margins of less than 2 cm and who were treated with adjuvant postoperative radiation had a local recurrence rate of 7%. In the previous period, the local recurrence rate was 30% after wide resection and 66.6% after local excision. With a combination of modalities, limb salvage can be practiced currently in the majority of patients with extremity soft tissue sarcomas without any adverse effect on recurrence rates and survival.


Usefulness of fragmented QRS on a 12-lead electrocardiogram in acute coronary syndrome for predicting mortality.
Electrocardiographic signs of a non-ST elevation myocardial infarction (NSTEMI) are nonspecific, and therefore the diagnosis of NSTEMI during acute coronary syndromes (ACS) depends mainly on cardiac biomarker levels. Fragmented QRS (fQRS) represents myocardial conduction abnormalities due to myocardial infarction (MI) scars in patients with coronary artery disease. However, the time of appearance of fQRS during ACS has not been investigated. It was postulated that in patients with ACS, fQRS on 12-lead electrocardiography occurs within 48 hours of presentation with NSTEMI as well as ST elevation MI and that fQRS predicts mortality. Serial electrocardiograms from 896 patients with ACS (mean age 62 +/- 11 years, 98% men) who underwent cardiac catheterization were studied. Four hundred forty-one patients had MIs, including 337 patients with NSTEMIs, and 455 patients had unstable angina (the control group). Serial electrocardiograms were obtained every 6 to 8 hours during the first 24 hours after the diagnosis of MI and the next day (<48 hours). Fragmented QRS on 12-lead electrocardiography was defined by the presence of single or multiple notches in the R or S wave, without a typical bundle branch block, in > or =2 contiguous leads in 1 of the major coronary artery territories. Fragmented QRS developed in 224 patients (51%) in the MI group and only 17 (3.7%) in the control group (p <0.001). New Q waves developed in 122 (28%), 76 (23%), and 2 (0.4%) patients in the MI, NSTEMI, and control groups, respectively. The sensitivity values of fQRS for ST elevation MI and NSTEMI were 55% and 50%, respectively. The specificity of fQRS was 96%. Kaplan-Meier survival analysis revealed that patients with fQRS had significantly decreased times to death compared to those without fQRS. Fragmented QRS, T-wave inversion, and ST depression were independent predictors of mortality during a mean follow-up period of 34 +/- 16 months. In conclusion, fQRS on 12-lead electrocardiography is a moderately sensitive but highly specific sign for ST elevation MI and NSTEMI. Fragmented QRS is an independent predictor of mortality in patients with ACS.


Drug-Releasing Thermogel for Osteoarthritis Induction in an Animal Model.
The induction of disease states in animal models is an essential step in new drug discovery procedures. In this study, osteoarthritis (OA) was induced in a mouse model using a polypeptide thermogel-based sustained drug release system. Hydrophilic lactobionic acids and hydrophobic n-butyric acids were grafted onto ε-poly(l-lysine) to prepare a thermogelling polymer of ε-poly(l-lysine) grafted with lactobionic acid and butyric acid (PLLB). The gel modulus of PLLB is about 1000 Pa at 37 °C. Collagenase, which causes OA, was slowly released from the PLLB thermogel over two weeks. The PLLB formulation containing collagenases ranging from 1-10 units was intra-articularly injected into the knee of mice. OA mouse models with Osteoarthritis Research Society International (OARSI) grades of 3-6 were developed depending on the amounts of collagenase incorporated in the PLLB thermogel formulation. This study suggests that thermogel-based drug release formulations can be a precise tool for developing animal disease models in a dose-dependent manner.


Effects of valdecoxib in the treatment of chronic low back pain: results of a randomized, placebo-controlled trial.
Valdecoxib, a cyclooxygenase (COX)-2 specific inhibitor, is indicated for relief of the signs and symptoms of rheumatoid arthritis, osteoarthritis, and primary dysmenorrhea. Therapeutic doses of COX-2 specific inhibitors are as effective as nonspecific nonsteroidal anti-inflammatory drugs in reducing inflammatory pain while sparing the gastrointestinal and platelet toxicity associated with nonspecific COX-1 inhibition. The aim of this study was to assess the analgesic efficacy and tolerability of valdecoxib 40 mg/d compared with placebo in the treatment of chronic low back pain. This 4-week, prospective, randomized, double-blind placebo-controlled, parallel-group study was conducted at 37 centers across the United States and 5 centers in Canada. Patients aged > or =18 years with chronic low back pain in flare were enrolled. Patients were randomized to receive valdecoxib 40-mg/d or placebo tablets, once daily for 4 weeks. Patients rated low back pain intensity on a visual analog scale and completed the Roland-Morris Disability Questionnaire and the modified Brief Pain Inventory-Short Form (mBPI-SF) at each visit. Two hundred ninety-three patients were enrolled. The valdecoxib group comprised 148 patients (81 women, 67 men; mean [SD] age, 48.6 [13.3] years; mean [SD] body weight, 86.6 [20.9] kg), and the placebo group included 145 patients (85 women, 60 men; mean [SD] age, 48.7 [12.6] years; mean [SD] body weight, 85.6 [19.9] kg). Of the enrolled patients, 249 completed the study: 134 patients (91%) who received valdecoxib and 115 patients (79%) who received placebo. No statistically significant differences in patient baseline characteristics were noted between treatment groups, except in response to 1 mBPI-SF question; patients in the valdecoxib group reported significantly greater interference in relations with other people due to pain than did those in the placebo group (P = 0.048). Changes from baseline in low back pain intensity were significantly greater in valdecoxib-treated patients at each assessment (all, P < 0.001 vs placebo). Pain scores on the mBPI-SF indicated significantly greater pain relief with valdecoxib at each assessment (all, P < or = 0.014 vs placebo). Improvements in mean Roland-Morris Disability Questionnaire score with valdecoxib were significantly greater than with placebo at each assessment (all, P < or = 0.003). Although the overall incidence of adverse events (AEs) was significantly higher among patients receiving valdecoxib than those receiving placebo (35.1% vs 24.1%, respectively; P = 0.042), no significant differences were found between groups for the incidence of any individual AE. Most AEs (89% [77/87 total events]) were mild or moderate in severity. In this study of patients with chronic low back pain, valdecoxib 40 mg/d provided rapid relief (within 1 week) and consistent relief (over 4 weeks). In addition, significant improvement in function and decreased disability were found with valdecoxib compared with placebo.


Colloidal assembly of polydisperse particle blends during drying.
In this work, we synthesize a polydisperse aqueous colloidal system composed of small and large zwitterionic particles, as well as medium sized standard acrylic particles. By assembling these dispersions into films by drying, we show using atomic force microscopy (AFM) how their top surfaces can be mostly covered by zwitterionic groups for a wide range of evaporation rates. We probe underneath the top film surface using Fourier-transform infrared (FTIR) spectroscopy - attenuated total reflection (ATR), observing that the content in zwitterionic particles of the film upper layer increases for faster evaporation rates. We show how polydisperse systems hold great potential to overcome the evaporation rate dependence of size segregation processes in drying colloidal blends, and we provide further insights into the assembly mechanisms involved. Polydisperse blends enhance the robustness of such processes for application in coatings and other soft products where evaporation rate can not be tuned.


Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles.
In this study, Autographa californica multiple nucleopolyhedrovirus ac75 was functionally characterized. Ac75 has homologs in all sequenced genomes of alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. It was determined to encode a protein that is associated with the nucleocapsid of budded virus and with both envelope and nucleocapsids of occlusion-derived virus. Sf9 cells transfected by an ac75-knockout bacmid resulted in the infection being restricted to single cells. No budded virus were detected although viral DNA replication and late gene expression were unaffected. Electron microscopy revealed that the virogenic stroma, nucleocapsids and occlusion bodies appeared normal in the cells transfected by an ac75-knockout bacmid. However, the nucleocapsids were unenveloped, the occlusion bodies did not contain any virions or nucleocapsids, and no nucleocapsids were found outside the nucleus or spanning the nuclear membrane. In addition, de novo intranuclear membrane microvesicles that are the precursor of occlusion-derived virus envelopes were absent in the nuclei of transfected cells. Confocal microscopy showed that AC75 protein appeared in the cytoplasm as early as 6 hours post infection. It localized to the ring zone at the periphery of the nucleus from 15 to 24 hours post infection and demonstrated light blocky cloud-like distribution in the center of the nucleus. AC75 was found to co-immunoprecipitate with BV and ODV associated envelope protein ODV-E25. The data from this study suggest that ac75 is essential for induction of the intranuclear membrane microvesicles, it appears to be required for the intranuclear envelopment of nucleocapsids, and is also essential for egress of nucleocapsids from the nuclei, in infected cells.


Regulation of canine jejunal transit.
The aim of this study was to determine what factors influence the direction of movement of canine jejunal chyme. In four dogs, pacing electrodes were implanted near each end of a 50-cm jejunal Vella loop, while recording electrodes and intraluminal pressure catheters were spaced along the loop. After recovery, the loop was perfused from either the proximal stoma (forward flow) or the distal stoma (reversed flow), and effluent was collected from the nonperfused stoma. The pacesetter potentials were paced electrically in a forward (aborad) or a reverse (orad) direction. During control conditions (forward flow-forward pacing), the mean transit time of liquids was 2.6 +/- 0.1 min (mean +/- SE) and the static volume of the loop was 8.8 +/- 0.3 ml. Reversing both direction of flow and direction of pacesetter potential propagation slowed transit (4.4 +/- 0.4 min; P less than 0.05) and increased loop volume (16.0 +/- 1.0 ml; P less than 0.01). Reversing flow with forward pacing resulted in even slower transit (7.5 +/- 1.2 min; P less than 0.05), maintained a large volume (33 +/- 10 ml), and increased basal pressure in the loop from -1.2 +/- 1.7 (control) to 2.3 +/- 1.6 mmHg (P less than 0.05). These observations suggest that the direction of propagation of jejunal pacesetter potentials influences the direction of movement of jejunal chyme but that other factors have a role as well.


Engineering complex muscle-tissue interfaces through microfabrication.
Skeletal muscle is a tissue with a complex and hierarchical architecture that influences its functional properties. In order to exert its contractile function, muscle tissue is connected to neural, vascular and connective compartments, comprising finely structured interfaces which are orchestrated by multiple signalling pathways. Pathological conditions such as dystrophies and trauma, or physiological situations such as exercise and aging, modify the architectural organization of these structures, hence affecting muscle functionality. To overcome current limitations of in vivo and standard in vitro models, microfluidics and biofabrication techniques have been applied to better reproduce the microarchitecture and physicochemical environment of human skeletal muscle tissue. In the present review, we aim to critically discuss the role of those techniques, taken individually or in combination, in the generation of models that mimic the complex interfaces between muscle tissue and neural/vascular/tendon compartments. The exploitation of either microfluidics or biofabrication to model different muscle interfaces has led to the development of constructs with an improved spatial organization, thus presenting a better functionality as compared to standard models. However, the achievement of models replicating muscle-tissue interfaces with adequate architecture, presence of fundamental proteins and recapitulation of signalling pathways is still far from being achieved. Increased integration between microfluidics and biofabrication, providing the possibility to pattern cells in predetermined structures with higher resolution, will help to reproduce the hierarchical and heterogeneous structure of skeletal muscle interfaces. Such strategies will further improve the functionality of these techniques, providing a key contribution towards the study of skeletal muscle functions in physiology and pathology.


Comparison of echocardiographic measurements in elite and nonelite Arabian endurance horses.
To determine whether echocardiographic variables differed between successful (elite) and less successful (nonelite) Arabian endurance horses. 34 Arabian horses that competed in endurance racing. Horses were assigned to either an elite or nonelite group on the basis of results of a previous competition, and a standardized echocardiographic examination was performed on each horse within 1 to 4 weeks after that competition. Multivariable logistic regression with backward stepwise elimination was used to create a prediction model for the determination of horse status (elite or nonelite) as a function of the measured echocardiographic variables. The elite and nonelite groups consisted of 23 and 11 horses, respectively. One horse in the nonelite group had a frequent ventricular dysrhythmia that could have negatively affected its performance and rider's safety, whereas none of the horses in the elite group had remarkable cardiac abnormalities. The left ventricular internal diameter during systole and diastole and left ventricular mass and stroke volume were significantly greater for horses in the elite group, compared with those for horses in the nonelite group. The final logistic regression model correctly predicted the horse status for all of the horses in the elite group and 8 of 11 horses in the nonelite group. Results indicated that heart size was significantly associated with performance for Arabian endurance horses in a manner similar to findings for Thoroughbred and Standardbred racehorses in active competition.


Ciclopirox nail lacquer for the treatment of onychomycosis: an open non-comparative study.
Onychomycosis is a relatively common disease accounting for up to 50% of all nail disorders. Topical treatment, although less effective than systemic, is usually preferred by patients. Topical antifungal nail lacquers have been formulated to provide better delivery of the antifungal agent to the nail unit. The purpose of this research is to evaluate the efficacy and safety of ciclopirox nail lacquer in the treatment of onychomycosis. Patients suffering from distal and lateral subungual toenail onychomycosis (DLSO) and lateral subungual onychomycosis (LSO) were treated by ciclopirox nail lacquer once daily for 9 months. Every week the nail lacquer was removed using acetone. Clinical nail status, KOH examination and mycological culture were recorded by the same investigator at 0, 3, 6 and 9 months. Thirty-six patients completed the 9-month regimen. Trichophyton rubrum was the most common pathogen. At the end of the study, good improvement to complete cure was observed in 13 patients (36%), 12 patients showed only mild to moderate improvement and 11 patients (31%) had no clinical improvement. No adverse effects were noted throughout the treatment period. Ciclopirox nail lacquer seems to be slightly more effective than other topical modalities and could be used in patients who cannot or do not want systemic treatment.


PAI-1 Regulation of TGF-β1-induced Alveolar Type II Cell Senescence, SASP Secretion, and SASP-mediated Activation of Alveolar Macrophages.
Senescence of alveolar type II (ATII) cells, progenitors of the alveolar epithelium, is a pathological feature and contributes importantly to the pathogenesis of idiopathic pulmonary fibrosis. Despite recognition of the importance of ATII cell senescence in idiopathic pulmonary fibrosis pathogenesis, how ATII cell senescence is regulated and how senescent ATII cells contribute to lung fibrogenesis remain unclear. In this study, we show that TGF-β1 (transforming growth factor-β1), a most ubiquitous and potent profibrotic cytokine, induces plasminogen activator inhibitor-1 (PAI-1), a cell senescence and fibrosis mediator, and p16 as well as senescence, but not apoptosis, in primary mouse ATII cells. We also found that senescent ATII cells secrete various cytokines and chemokines, including IL-4 and IL-13, which stimulate the expression of genes associated with a profibrotic phenotype in alveolar macrophages. Similar responses were also observed in TGF-β1-treated rat ATII (L2) and rat macrophage NR8383 cells. Deletion of PAI-1 or inhibition of PAI-1 activity with a small molecule PAI-1 inhibitor, however, blocks TGF-β1-induced senescence as well as a senescence-associated secretory phenotype in ATII and L2 cells and, consequently, the stimulatory effects of the conditioned medium from senescent ATII/L2 cells on macrophages. Moreover, we show that silencing p16 ameliorates PAI-1 protein-induced ATII cell senescence and secretion of profibrotic mediators. Our data suggest that PAI-1 mediates TGF-β1-induced ATII cell senescence and secretion of profibrotic mediators through inducing p16, and they also suggest that senescent ATII cells contribute to lung fibrogenesis in part by activating alveolar macrophages through secreting profibrotic and proinflammatory mediators.


Sex hormones and modulation of immunity against leishmaniasis.
Sex-associated hormones such as estradiol, testosterone and progesterone have all been shown to modulate immune responses, which can result in differential disease outcomes between males and females, as well as between pregnant and nonpregnant females. Most parasitic diseases, including leishmaniasis, usually result in more severe disease in males compared with females. This review highlights our current knowledge concerning the role of sex hormones in modulating leishmaniasis in both clinical settings and experimental disease models.


Ethanol-induced osmolality changes and lymphocyte proliferation.
While there have been reports on the in vitro effects of ethanol on mitogen-stimulated lymphocyte proliferation, little attention has been paid to whether these effects are mediated by the ethanol molecule per se or the associated hyperosmolality. There is controversy whether ethanol at high concentrations inhibits or has no effect on mitogen-stimulated proliferation of T cells. In this study we sought to distinguish between effects due to ethanol and those related to increased osmolality. Rat splenocytes were cultured in vitro in hypertonic (320 mOsm) or isotonic (320 mOsm) ethanol solutions (up to 400 mg/dL), or in corresponding hypertonic solutions without ethanol. Proliferative responses to the B cell mitogen lipopolysaccharide and the T cell mitogen concanavalin A were measured by mitochondrial dimethylthiazole diphenyl tetrazolium metabolism and [(3) H]-thymidine incorporation into DNA. B cell response was not significantly affected by ethanol under any conditions. By contrast, T cell proliferative responses were significantly inhibited by isotonic ethanol solutions or hypertonic solutions in the absence of ethanol, but not by hypertonic ethanol solutions. These results indicate that, while high osmolality is normally inhibitory to lymphocyte function, the increase in osmolality caused by ethanol is protective against the otherwise detrimental effects of ethanol.


[Diffusion-weighted magnetic resonance tomography--a new instrument in the diagnosis of Creutzfeldt-Jakob disease].
Creutzfeldt-Jakob's disease (CJD) is characterised by rapidly progressive dementia, ataxia, myoclonus, and several other neurological deficits. It generally affects older adults and occurs in sporadic, genetic and iatrogenic forms. Death occurs usually within one year after onset of the disease. The diagnosis is based on clinical criteria, neurophysiological and radiological findings, and confirmed by postmortal histopathology. During the last two years several cases of CJD have been reported with diffusion-weighted magnetic resonance imaging (MR) abnormalities, represented by increased signal intensity indicating reduced diffusion in basal ganglia and/or cortex cerebri. These abnormalities seem to be characteristic of CJD. We report a case of CJD in a 54-year-old woman who developed vertigo, nystagmus, ataxia, myoclonus, and dementia over a period of eight months. Diffusion-weighted magnetic resonance imaging showed increased signal intensity in corpus striatum and gyrus cinguli. The diagnosis was postmortally confirmed with histopathology.


Cytological features of non-small cell carcinomas of the lung in fine needle aspirates.
Fifty eight lung tumours were typed according to the second World Health Organization histological classification and compared with the cytological appearances obtained by fine needle aspiration in a total of 47 primary non-small cell carcinomas. The presence of glands, cell balls, branching or papillary structures, cylindrical cells and nuclear grooving were major diagnostic indicators for adenocarcinoma. Cytoplasmic macrovacuoles were more common in adenocarcinomas (69%) than in squamous (37%) and large cell (50%) carcinomas. Two or more of these features were combined in all well and moderately differentiated adenocarcinomas and in 67% of poorly differentiated adenocarcinoma. The major cytological indicators for squamous carcinomas were the presence of keratin and eosinophilic spindle cells with glassy or laminated cytoplasm. Granular cytoplasm was not specific for any histological type. A combination of the major features for both adeno- and squamous carcinoma was present in 58% of adeno-squamous carcinomas, including some poorly differentiated types. Correct typing could be obtained in almost all the well and moderately differentiated carcinomas and in about two thirds of the poorly differentiated tumours using FNA, provided that combinations and not individual variables are considered.


Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.
Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.


Structural and functional characterization of HLA-DR molecules circulating in the serum.
HLA molecules are highly polymorphic glycoproteins with a single binding site for immunogenic peptides. The complex formed by HLA-DR molecules and peptides is the entity specifically recognized by the antigen receptor of CD4+ helper T lymphocytes. This biological function has been linked to the constitutive cell surface expression of HLA molecules on antigen-presenting cells which provide immunogenic peptides through denaturation or fragmentation of antigen. Here, we report that HLA-DR molecules can be identified in a soluble form circulating in the serum. Biochemical characterization and recognition of such soluble HLA-DR molecules by a monoclonal antibody reactive to conformational determinants on the alpha-beta HLA complex indicates that they are circulating as intact heterodimers. Upon activation, human T cells and B cells release soluble HLA-DR molecules. The expression and secretion of HLA-DR by T lymphocytes is greatly enhanced in the presence of macrophages indicating that the production of HLA-DR molecules might be regulated and controlled through factors derived from antigen-presenting cells. An alloreactive human T cell clone can be stimulated to proliferate in response to soluble HLA-DR molecules purified and enriched from serum suggesting that the soluble form of the natural ligand of the T cell receptor might have immunoregulatory functions.


Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids.
A soluble aldehyde dehydrogenase (EC 22.214.171.124) was partially purified from Rhizobium japonicum bacteroids and from free-living R. japonicum 61A76. The enzyme was activated by NAD+, NADH, and dithiothreitol, and it reduced NAD(P)+. Acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and succinic semialdehyde were substrates. The Km for straight-chain aldehydes decreased with increasing carbon chain length. The aldehyde dehydrogenase was inhibited by 6-cyanopurine, but not by metronidazole. These compounds inhibited acetylene reduction, but not respiration, by isolated bacteroids.


Autoinhibition of bacteriophage T4 Mre11 by its C-terminal domain.
Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double strand breaks. In the bacteriophage T4, Rad50 (gene product 46) enhances the nuclease activity of Mre11 (gene product 47), and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross-activation of the MR complex has been elusive. Various crystal structures of the MR complex display limited protein-protein interfaces that mainly exist between the C terminus of Mre11 and the coiled-coil domain of Rad50. To test the role of the C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed a series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of the highly acidic flexible linker that lies between RBD and the main body of Mre11 increases the nuclease activity of Mre11 by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of the MR complex when combined with deletion of RBD. Nuclease activity kinetics indicate that Rad50 association and deletion of the C terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil.


Detection of Drug-Drug Interactions Inducing Acute Kidney Injury by Electronic Health Records Mining.
While risk of acute kidney injury (AKI) is a well documented adverse effect of some drugs, few studies have assessed the relationship between drug-drug interactions (DDIs) and AKI. Our objective was to develop an algorithm capable of detecting potential signals on this relationship by retrospectively mining data from electronic health records. Data were extracted from the clinical data warehouse (CDW) of the Hôpital Européen Georges Pompidou (HEGP). AKI was defined as the first level of the RIFLE criteria, that is, an increase ≥50 % of creatinine basis. Algorithm accuracy was tested on 20 single drugs, 10 nephrotoxic and 10 non-nephrotoxic. We then tested 45 pairs of non-nephrotoxic drugs, among the most prescribed at our hospital and representing distinct pharmacological classes for DDIs. Sensitivity and specificity were 50 % [95 % confidence interval (CI) 23.66-76.34] and 90 % (95 % CI 59.58-98.21), respectively, for single drugs. Our algorithm confirmed a previously identified signal concerning clarithromycin and calcium-channel blockers (unadjusted odds ratio (ORu) 2.92; 95 % CI 1.11-7.69, p = 0.04). Among the 45 drug pairs investigated, we identified a signal concerning 55 patients in association with bromazepam and hydroxyzine (ORu 1.66; 95 % CI 1.23-2.23). This signal was not confirmed after a chart review. Even so, AKI and co-prescription were confirmed for 96 % (95 % CI 88-99) and 88 % (95 % CI 76-94) of these patients, respectively. Data mining techniques on CDW can foster the detection of adverse drug reactions when drugs are used alone or in combination.