Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins

A molecular test for Alzheimer's disease could lead to better treatment and therapies. We found 18 signaling proteins in blood plasma that can be used to classify blinded samples from Alzheimer's and control subjects with close to 90% accuracy and to identify patients who had mild cognitive impairment that progressed to Alzheimer's disease 2-6 years later. Biological analysis of the 18 proteins points to systemic dysregulation of hematopoiesis, immune responses, apoptosis and neuronal support in presymptomatic Alzheimer's disease.     

Complementation of placental defects and embryonic lethality by trophoblast-specific lentiviral gene transfer

Placental dysfunction underlies many complications during pregnancy, and better understanding of gene function during placentation could have considerable clinical relevance. However, the lack of a facile method for placenta-specific gene manipulation has hampered investigation of placental organogenesis and the treatment of placental dysfunction. We showed previously that transduction of fertilized mouse eggs with lentiviral vectors leads to transgene expression in both the fetus and the placenta. Here we report placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts after removal of the zona pellucida. All of the placentas analyzed, but none of the fetuses, were transgenic. Application of this method substantially rescued mice deficient in Ets2, Mapk14 (also known as p38alpha) and Mapk1 (also known as Erk2) from embryonic lethality caused by placental defects. Ectopic expression of Mapk11 also complemented Mapk14 deficiency during placentation.     

Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid involved in diverse biological processes, is generated by sphingosine kinase (SphK) and acts via intracellular and/or extracellular mechanisms. We used biochemical, pharmacological, and physiological approaches to investigate in rat myometrium the contractile effect of exogenous S1P and the possible contribution of SphK in endothelin-1 (ET-1)-mediated contraction. S1P stimulated uterine contractility (EC₅₀ = 1 µM and maximal response = 5 µM) by a pertussis toxin-insensitive and a phospholipse C (PLC)-independent pathway. Phosphorylated FTY720, which interacts with all S1P receptors, except S1P₂ receptors, failed to mimic S1P contractile response, indicating that the effects of S1P involved S1P₂ receptors that are expressed in myometrium. Contraction mediated by S1P and ET-1 required extracellular calcium and Rho kinase activation. Inhibition of SphK reduced ET-1-mediated contraction. ET-1, via ET_A receptors coupled to pertussis toxin-insensitive G proteins, stimulated SphK1 activity and induced its translocation to the membranes. Myometrial contraction triggered by ET-1 is consecutive to the sequential activation of PLC, protein kinase C, SphK1 and Rho kinase. Prolonged exposure of the myometrium to S1P downregulated S1P₂ receptors and abolished the contraction induced by exogenous S1P. However, in these conditions, the tension triggered by ET-1 was not reduced, indicating that SphK activated by ET-1 contributed to its contractile effect via a S1P₂ receptor-independent process. Our findings demonstrated that exogenous S1P and SphK activity regulated myometrial contraction and may be of physiological relevance in the regulation of uterine motility during gestation and parturition. uterus; contraction     

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Induction of multiple cytokine secretion from RAW264.7 cells by alginate oligosaccharides

We investigated the cytokine-inducing activities of guluronate (G3-G6) and mannuronate (M3-M6) oligomers on RAW264.7 cells with the Bio-Plex assay system. Relatively high levels of tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted (RANTES), granulocyte macrophage (GM)-CSF, and eotaxin were induced by alginate oligomers to different extents depending on the oligomer structures, and low but significant levels of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-9, and IL-13 were also induced. Throughout all cytokines tested, M-oligomers tended to be more potent than G-oligomers in terms of cytokine induction, and this tendency was evident in differences between G3 and M3.     

Clinical Features and Mortality of COVID-19-Associated Mucormycosis: A Systematic Review and Meta-Analysis

The recent increase of COVID-19-associated mucormycosis (CAM) has been commanding global attention. However, basic epidemiologic characteristics have not firmly been established. In this systematic review and meta-analysis, we sought to determine the clinical manifestations, potential risk factors, and outcomes of CAM. Observational studies reporting CAM were searched with PubMed and EMBASE databases in January 2022. We collected data on comorbidities and treatment for COVID-19, and performed a one-group meta-analysis on the frequency of orbital exenteration procedure and mortality of CAM using a random-effect model. Fifty-one observational studies, including a total of 2,312 patients with proven CAM, were identified. Among the 51 studies, 37 were conducted in India, 8 in Egypt, and 6 in other countries. The most common comorbidity was diabetes mellitus (82%). While 57% required oxygenation, 77% received systemic corticosteroids. Among CAM, 97% were rhino-orbital-cerebral (ROCM), and 2.7% were pulmonary mucormycosis. Usual presentations were headache (54%), periorbital swelling/pain (53%), facial swelling/pain (43%), ophthalmoplegia (42%), proptosis (41%), and nasal discharge/congestion (36%). Regarding the outcomes, orbital exenteration was performed in 17% (95% CI: 12–21%, I²?=?83%) of the COVID-19-associated ROCM patients. The mortality of CAM was 29% (95% CI; 22–36%, I²?=?92%). In conclusion, this systematic review and meta-analysis indicated that the most prevalent type of CAM was ROCM, and most CAM patients had diabetes mellitus and received systemic glucocorticoids. Clinicians in the endemic areas should have a high index of suspicion for this invasive fungal complication of COVID-19 when a diabetic patient who received high-dose systemic glucocorticoids developed rhino-orbital symptoms.

     

Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His–Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser²⁵⁰, which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser²⁵⁰ substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser²⁵⁰ phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys²⁶⁸ in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.