Characterization of a Calmodulin Antiserum by its Reactions with Fragments of the Calmodulin Molecule

Abstract

A high affinity antibody, specific to the calcium-free form af calmodulin, which had previously been developed using N-acetyl-muramyl-L-alanyl-D-isoglutamine-calmodulin conjugate as an immunogen, was tested for cross-reactivity with tryptic fragments of calmodulin (CaM_1-77, CaM_1-90, CaM_78-149, and CaM_106-149) as well as with synthetic peptides corresponding to the 1st, 2nd, and 3rd calcium binding loop of calmodulin. The results showed that the antigenic determinant involves a special conformation of amino acid residues 90-106 in the 3rd calcium-binding domain.     

Diagnostic specificity of the African swine fever virus antibody detection enzyme‐linked immunosorbent assay in feral and domestic pigs in the United States

African swine fever (ASF) is a highly contagious haemorrhagic disease of pigs that has the potential to cause mortality nearing 100% in naïve animals. While an outbreak of ASF in the United States’ pig population (domestic and feral) has never been reported, an introduction of the disease has the potential to cause devastation to the pork industry and food security. During the recovery phase of an outbreak, an antibody detection diagnostic assay would be required to prove freedom of disease within the previously infected zone and eventually nationwide. Animals surviving an ASF infection would be considered carriers and could be identified through the persistence of ASF viral antibodies. These antibodies would demonstrate exposure to the disease and not vaccination, as there is no ASF vaccine available. A well‐established commercial enzyme‐linked immunosorbent assay (ELISA) detects antibodies against ASF virus (ASFV), but the diagnostic specificity of the assay had not been determined using serum samples from the pig population of the United States. This study describes an evaluation of the World Organization for Animal Health (OIE)‐recommended Ingezim PPA COMPAC ELISA using a comprehensive cohort (n = 1791) of samples collected in the United States. The diagnostic specificity of the assay was determined to be 99.4% (95% confidence interval (CI): [98.9, 99.7]). The result of this study fills a gap in understanding the performance of the Ingezim PPA COMPAC ELISA in the ASF naïve pig population of the United States.     

Applause sign: screening utility for dementia and cognitive impairment

Objective: To examine the diagnostic utility of applause sign scores for the diagnosis of dementia and mild cognitive impairment. Methods: Consecutive unselected new outpatient referrals to a dedicated cognitive disorders clinic over a 12-month period were administered the clapping test. Criterion diagnosis was by usual clinic assessment using standard diagnostic criteria, blind to applause sign score. Results: Applause sign scores differed significantly (p < 0.001) between diagnostic groups (dementia, mild cognitive impairment, subjective memory complaint) and did not correlate with other cognitive screening instrument scores. Nearly three-quarters of those with an abnormal score had cognitive impairment. Applause sign score was specific but not sensitive for a diagnosis of cognitive impairment. Conclusion: The applause sign supports a diagnosis of dementia or cognitive impairment in high prevalence settings and may be useful in conjunction with other cognitive screening tests.     

Detection of Mycobacterium tuberculosis growth by direct microscopic observation has a lower cost,higher sensitivity and higher diagnostic value than liquid and solid media cultures

Background: Culture is needed to confirm tuberculosis but results are generally obtained after several weeks. Objectives: We compared a direct microscopic observation technique for detection of mycobacterial culture positivity (MODS) with the classic solid and MB/BacT cultures in terms of sensitivity, contamination rate, speed and cost on 488 samples. Results: The sensitivity of the MODS technique - 99,2% (162 positive samples) was higher than MB/BacT 78,4% (125 positive samples) and solid culture 69,6% (113 positive samples) P < 0.005 for all comparisons. The median times to positivity were 21, 13.3 and 3 days on solid media, B/BacT and MODS respectively. Conclusions: The MODS technique is faster and more sensitive than both solid media and MB/BacT culture.     

Characterization of UBO-QIC as a Gαq inhibitor in platelets

Gα_q plays an important role in platelet activation by agonists such as thrombin, adenosine diphosphate (ADP) and thromboxane. The significance of Gα_q signaling in platelets was established using YM254890, a Gα_q/11-specific inhibitor and Gα_q knockout murine platelets. However, YM-254890 is no longer available for investigators and there is a need to characterize other Gα_q inhibitors. The aim of this study is to characterize the specificity of a compound, {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)} (UBO-QIC), as a Gα_q inhibitor in platelets. Human platelets treated with UBO-QIC showed a concentration-dependent inhibition of platelet aggregation and secretion by protease-activated receptors (PAR) agonists, U46619 and ADP. UBO-QIC also abolished Gα_q pathway signaling events such as calcium mobilization and pleckstrin phosphorylation. UBO-QIC had no nonspecific effects on the Gα_12/13 pathway since platelet shape change was intact in Gα_q knockout murine platelets stimulated with PAR agonists in the presence of the inhibitor. In addition, UBO-QIC-treated platelets did not affect collagen-related peptide-induced platelet activation suggesting that this inhibitor had no non-specific effects on the GPVI pathway. Furthermore, Akt phosphorylation downstream of the Gα_i and Gα_z pathways, and vasodilator-stimulated phosphoprotein phosphorylation downstream of the Gα_s pathway were not inhibited in UBO-QIC-treated platelets. UBO-QIC is a specific inhibitor for Gα_q, which can be a useful tool for investigating Gα_q-coupled receptor signaling pathways in platelets.