Flow Cytometric Test with Eosin-5-Maleimide for a Diagnosis of Hereditary Spherocytosis in a Newborn

A term male newborn born to a mother who had hereditary spherocytosis presented with neonatal jaundice at 20 hours of life. Complete blood count showed hemoglobin 17.1 g/dL, MCV 104.2 fL, MCH 32.9 pg, and MCHC 31.6 g/dL. The patient had indirect hyperbilirubinemia requiring phototherapy. The maximum total bilirubin level was 12.15 mg/dL at 20 hours of life. Peripheral blood smear revealed spherocytes, crenated red cells, and polychromasia. A flow cytometric test with eosin-5-maleimide- (EMA-) labeled RBC was performed in the patient and parents. The fluorescence histograms of EMA-labeled RBC from the patient and mother were shifted to the left, and the fluorescence ratio when compared with normal was 0.69 and 0.84, respectively. The flow cytometric test with EMA is useful in supporting the diagnosis of hereditary spherocytosis during newborn period

The efficacy of alendronate for the treatment of thalassemia-associated osteoporosis: a randomized controlled trial

BackgroundWith adequate blood transfusion and iron chelation, thalassemia patients have a longer life expectancy and experience long-term metabolic complications, including osteoporosis, fractures, and bone pain. Alendronate, an oral bisphosphonate, is currently used to treat various types of osteoporosis. However, the efficacy for the treatment of thalassemia-associated osteoporosis remains unclear.MethodsWe conducted a randomized controlled trial to evaluate the efficacy of alendronate for the treatment of osteoporosis in thalassemia patients. Patients were included if they were males (18–50 years) or premenopausal females with low bone mineral density (BMD) (Z-score < -2.0 SD) or positive vertebral deformities from vertebral fracture analysis (VFA). Stratified randomization was performed according to sex and transfusion status. Patients were 1:1 allocated to receive once weekly alendronate 70 mg orally or placebo for a total duration of 12 months. BMD and VFA were re-evaluated at 12 months. Markers of bone resorption (C-terminal crosslinking telopeptide of type I collagen; CTX) and bone formation (Procollagen type I N-terminal propeptide; P1NP), and pain scores were measured at baseline, 6 months, and 12 months. The primary outcome was the change of BMD. The secondary endpoints were changes in bone turnover markers (BTM) and pain scores.ResultsA total of 51 patients received the study drug, 28 patients were assigned to receive alendronate and 23 patients to receive placebo. At 12 months, patients in the alendronate group had significant improvement of BMD at L1-L4 compared to their baseline (0.72 ± 0.11 vs 0.69 ± 0.11 g/cm2, p = 0.004), while there was no change in the placebo group (0.69 ± 0.09 vs 0.70 ± 0.06 g/cm2, p = 0.814). There was no significant change of BMD at femoral neck in both groups. Serum BTMs were significantly decreased among patients receiving alendronate at 6 and 12 months. The mean back pain score was significantly reduced compared to the baseline in both groups (p = 0.003). Side effects were rarely found and led to a discontinuation of the study drug in 1 patient (grade 3 fatigue).ConclusionAlendronate 70 mg orally once weekly for 12 months effectively improves BMD at L-spine, reduces serum BTMs, and alleviates back pain in thalassemia patients with osteoporosis. The treatment was well tolerated and had a good safety profile

Versican contributes to ligament formation of knee joints

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments’ formation and that its depletion affects joint function in later ages

Additional file 1: of Effects of sesamin on primary human synovial fibroblasts and SW982 cell line induced by tumor necrosis factor-alpha as a synovitis-like model

LDH released from hSF and SW982 cells. Cell viability testing was performed by LDH measurement in culture medium. a. LDH released from hSF cells under TNF-α alone, sesamin alone or combination of TNF-α and sesamin. b. LDH released from SW982 cells under TNF-α alone, sesamin alone or combination of TNF-α and sesamin. Values are presented as mean ± SEM (n = 3). (PNG 135 kb

Alpha-helicoidal HEAT-like repeat proteins (alpha Rep) selected as interactors of HIV-1 nucleocapsid negatively interfere with viral genome packaging and virus maturation

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (alpha Rep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of alpha Rep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong alpha Rep binders were isolated from the screen, alpha Rep4E3 (32 kDa; 7 internal repeats) and alpha Rep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both alpha Rep4E3 and alpha Rep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: alpha Rep4E3 blocked the viral genome packaging, whereas alpha Rep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing alpha Rep9A8 acquired long-term resistance to HIV-1, implying that alpha Rep proteins can act as antiviral restriction-like factors

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1

Background: Ank GAG 1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of Ank GAG 1D4 would potentially enhance the Ank GAG 1D4-mediated antiviral activity. Objective: To augment the affinity of Ank GAG 1D4 scaffold towards its CA target, through computational predictions and experimental designs. Method: Three dimensional structure of the binary complex formed by Ank GAG 1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified Ank GAG 1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI). Results: Tyrosine at position 56 (Y56) in Ank GAG 1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in Ank GAG 1D4-S45Y mutant, with no alteration of the target specificity. Conclusions: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of Ank GAG 1D4 ankyrin for its CA target. Ank GAG 1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future