Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen

BACKGROUND: The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti-KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti-KEL1 reagents. STUDY DESIGN AND METHODS: Kell genotyping methods, including KEL exon 6 direct sequencing, were applied. KEL cDNA from D1 was sequenced. Flow cytometry was used to assess KEL1 and KEL2 RBC expression. RESULTS: RBCs from the donor, her mother, and an unrelated donor gave weak or negative reactions with some anti-KEL1 reagents. Other Kell-system antigens appeared normal. The three individuals were homozygous for KEL C578 (KEL*2) but heterozygous for a 577A>T transversion, encoding Ser193. They appeared to be KEL*2 homozygotes by routine genotyping methods. Flow cytometry revealed weak KEL1 expression and normal KEL2, similar to that of KEL*2 homozygotes. CONCLUSION: Ser193 in the Kell glycoprotein appears to result in expression of abnormal KEL1, in addition to KEL2. The mutation is not detected by routine Kell genotyping methods and, because of unpredicted KEL1 expression, could lead to a misdiagnosis.

A NOVEL AND SIMPLE RULE OF THUMB FOR MULTIPLICITY CONTROL IN EQUIVALENCE TESTING USING TWO ONE-SIDED TESTS

Equivalence testing is growing in use in scientific research outside of its traditional role in the drug approval process. Largely due to its ease of use and recommendation from the United States Food and Drug Administration guidance, the most common statistical method for testing (bio)equivalence is the two one-sided tests procedure (TOST). Like classical point-null hypothesis testing, TOST is subject to multiplicity concerns as more comparisons are made. In this manuscript, a condition that bounds the family-wise error rate (FWER) using TOST is given. This condition then leads to a simple solution for controlling the FWER. Specifically, we demonstrate that if all pairwise comparisons of k independent groups are being evaluated for equivalence, then simply scaling the nominal Type I error rate down by (k - 1) is sufficient to maintain the family-wise error rate at the desired value or less. The resulting rule is much less conservative than the equally simple Bonferroni correction. An example of equivalence testing in a non drug-development setting is given.

Design and implementation of a 3D computer game controller using inertial MEMS sensors

Though 3D computer graphics has seen tremendous advancement in the past two decades, most available mechanisms for computer interaction in 3D are high cost and targeted for industry and virtual reality applications. Recent advances in Micro-Electro-Mechanical-System (MEMS) devices have brought forth a variety of new low-cost, low-power, miniature sensors with high accuracy, which are well suited for hand-held devices. In this work a novel design for a 3D computer game controller using inertial sensors is proposed, and a prototype device based on this design is implemented. The design incorporates MEMS accelerometers and gyroscopes from Analog Devices to measure the three components of the acceleration and angular velocity. From these sensor readings, the position and orientation of the hand-held compartment can be calculated using numerical methods. The implemented prototype is utilizes a USB 2.0 compliant interface for power and communication with the host system. A Microchip dsPIC microcontroller is used in the design. This microcontroller integrates the analog to digital converters, the program memory flash, as well as the core processor, on a single integrated circuit. A PC running Microsoft Windows operating system is used as the host machine. Prototype firmware for the microcontroller is developed and tested to establish the communication between the design and the host, and perform the data acquisition and initial filtering of the sensor data. A PC front-end application with a graphical interface is developed to communicate with the device, and allow real-time visualization of the acquired data.