Development and validation of an ultra-performance liquid chromatography with tandem mass spectrometry method for determination of soluble repulsive guidance molecule A in human serum and cerebrospinal fluid.

Repulsive guidance molecule A (RGMa) is upregulated in neurodegenerative diseases. To assess RGMa levels in human serum and cerebrospinal fluid (CSF), a quantification method was developed and validated according to ICH M10 guideline. Sample preparation consisted of immunoprecipitation (IP, only for serum), digestion and purification followed by MS.

CTLA4-Ig-Based Bifunctional Costimulation Inhibitor Blocks CD28 and ICOS Signaling to Prevent T Cell Priming and Effector Function.

CTLA4-Ig/abatacept dampens activation of naive T cells by blocking costimulation via CD28. It is an approved drug for rheumatoid arthritis but failed to deliver efficacy in a number of other autoimmune diseases. One explanation is that activated T cells rely less on CD28 signaling and use alternate coreceptors for effector function.

CD40/anti-CD40 antibody complexes which illustrate agonist and antagonist structural switches.

Background: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases.

Small Molecule IL-36γ Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis.

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate.

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation.

Structural and functional conservation of Mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin.

GroEL is a group I chaperonin that facilitates protein folding and prevents protein aggregation in the bacterial cytosol. Mycobacteria are unusual in encoding two or more copies of GroEL in their genome. While GroEL2 is essential for viability and likely functions as the general housekeeping chaperonin, GroEL1 is dispensable, but its structure and function remain unclear.

The M-domain controls Hsp104 protein remodeling activity in an Hsp70/Hsp40-dependent manner.

Yeast Hsp104 is a ring-forming ATP-dependent protein disaggregase that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. Both upstream and downstream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to Hsp104 protein remodeling activity. Hsp104 is a multidomain protein that possesses an N-terminal domain, an M-domain, and two tandem AAA(+) domains.

CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2.

Crystallization and preliminary X-ray crystallographic analysis of a GroEL1 fragment from Mycobacterium tuberculosis H37Rv.

Full-length GroEL1 from Mycobacterium tuberculosis H37Rv was cloned, overexpressed and purified. Crystals were obtained by the hanging-drop vapor-diffusion method and contained a 23 kDa GroEL1 fragment. A complete native data set was collected from a single frozen crystal that belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 75.

Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system.

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system.

Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously.

A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine.

A cytochrome P450 and an iron-sulfur protein, whose expression was specifically induced during growth of Mycobacterium sp. strain HE5 on morpholine as the sole source of carbon, nitrogen, and energy were purified to apparent homogeneity. Due to the lack of enzymatic activity, carbon monoxide difference spectra and determination of the acid-labile sulfur, respectively, were used to detect the proteins during purification.

High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp. strain HE5.

Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil. The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis. An identity of 99.

Outcome of double-blind, placebo-controlled food challenge tests in 107 children with atopic dermatitis.

Background And Objective: Little is known about late phase clinical reactions during oral food challenges and the value of specific IgE in terms of sensitivity and specificity.

Methods: We therefore analysed retrospectively the clinical outcome of 387 oral provocations during double-blind, placebo-controlled food challenge tests in 107 children with atopic dermatitis.

Results: Eighty-seven (81%) children showed a positive clinical reaction to at least one challenge.

The clinical workstation as a means of improving laboratory use.

Driven to make timely decisions, doctors may act with less than all of the relevant patient data. Sub-optimal use of clinical laboratory resources can result. Our clinicians' workstation (CWS) is meant to provide doctors and nurses ready access to laboratory results in a form that makes the data easy to review and use.

Closing the clinical laboratory testing loop with information technology.

Information technology can enhance the effectiveness of the part of the laboratory testing loop that occurs outside of the clinical laboratory. That external component involves the presentation of laboratory results and related information to physicians and the reception of physician requests for follow-up testing. Improvements made in the clinician-computer interface can conceivably enhance the quality of information transfer of this part of the testing loop and thereby improve the effectiveness of the entire loop.

A clinician's workstation for improving laboratory use. Integrated display of laboratory results.

Physicians are often forced to make decisions about the use of laboratory resources without adequate access to earlier results and related supporting information. Less than optimal use of the laboratory may result. The authors developed and deployed a clinical workstation meant to provide ready access to laboratory information that is presented in a format well-matched to the patient monitoring task.

Integrating legacy laboratory information systems into a client-server world: the University of Minnesota Clinical Workstation (CWS) project.

The development of an innovative clinical decision-support project such as the University of Minnesota's Clinical Workstation initiative mandates the use of modern client-server network architectures. Preexisting conventional laboratory information systems (LIS) cannot be quickly replaced with client-server equivalents because of the cost and relative unavailability of such systems. Thus, embedding strategies that effectively integrate legacy information systems are needed.

The deployment of a World Wide Web (W3) based medical information system.

The development of Web technologies has revolutionized information dissemination on the Internet. The University of Minnesota Hospital and Clinic's Web Clinical Information System (CIS) demonstrates the use of the Web as an infrastructure for deploying a medical information system at a fraction of the developmental cost of more traditional client server systems. This Web CIS has been deployed since December 1994.

Maternal time and the care of disabled children.

Caring for disabled children has become increasingly the responsibility of parents, even when the medical care is complex. To assess the time commitment required, 133 mothers of disabled children were asked to estimate by specific task categories the extra time required to care for the children. Total average daily care time was reported at 12 hours and 6 minutes, with 6 hours and 30 minutes consumed in "vigilant" tasks (i.

Determinants of home care nursing hours for technology-assisted children.

In the 1980s home care, in contrast to hospital care, was reported substantially to reduce costs for third-party payers who provided funding for technology-assisted children. Savings were realized primarily because parents substituted for nurses, eliminating or reducing those costs. Third-party payers' savings thus were directly related to the number of hours parents assumed care.

Self-concept of children and adolescents with cleft lip and/or palate.

The self-concept of 105 children (8 to 11 years) and adolescents (12 to 18 years) with cleft lip and/or palate (CLP) was studied using the Piers-Harris Children's Self-Concept Scale and selected demographic and medical variables. Results indicated that most (98%) of children had average or above average self-concept scores. Further analysis, however, demonstrated an interaction between age and gender: adolescent girls experienced a more negative self-concept in comparison to younger girls and adolescent boys experienced a more positive self-concept in comparison to younger boys.

Design and preliminary evaluation of an expert system for platelet request evaluation.

In spite of growing awareness of the potential risks associated with transfusion, the number of platelet units transfused in the United States continues to increase each year. There is a growing interest in ensuring that all transfusions are administered for appropriate reasons. Prospective review of requests for transfusions has been used to accomplish this goal.

ESPRE--expert system for platelet request evaluation.

The expert system for platelet request evaluation (ESPRE) is being developed to support independent learning and decision making regarding the use of platelet transfusions while physicians are actively engaged in clinical practice. The knowledge of transfusion medicine incorporated in ESPRE has been largely gathered from blood bank physicians responsible for determining the appropriateness of blood product administration. Knowledge acquisition methods have included structured and unstructured interviews with protocol analysis using real and fabricated transfusion cases and critiques of an expert system prototype's conclusions.