Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 Are Identified in Individuals with Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ∼12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH

Long-term follow-up and exploration of the mechanism of stromal vascular fraction gel in chronic wounds

Abstract Background Chronic refractory wounds easily relapse and seriously affect the patients’ quality of life. Previous studies have shown that stromal vascular fraction gel (SVF-gel) significantly promotes the early healing of chronic wounds; however, the mechanisms of SVF-gel function per se remain unclear, and a long-term follow-up is lacking. This study aims to explore the mechanisms of SVF-gel promoting the healing of chronic wounds and follow up the long-term efficacy of SVF-gel. Methods Autologous SVF-gel transplantation was performed in 20 patients with chronic wounds (from March 2016 to September 2019), and the size of the wound before and after SVF-gel transplantation was observed. The conditioned medium (CM) was harvested from SVF-gel under serum-free, serum-deprivation and 10% fetal bovine serum (FBS) microenvironment in vitro, respectively. The concentration of the growth factors in the two kinds of gel-CM was tested, and their effects on the proliferation and migration of human dermal fibroblasts (HDFs) were detected. Results All patients had 100% wound closure eventually, and the average time to complete closure was 28.3 ± 9.7 days. The time of follow-up ranged from 2 to 6 years, and there was no wound recurrence. Interestingly, the concentrations of epidermal growth factor and transforming growth factor β1 of the CM were higher in serum-free and serum-deprivation condition than in 10% FBS microenvironment (p < 0.05). Correspondingly, the proliferation and migration ability of HDFs treated with gel-CM from serum-free condition were stronger than those treated with gel-CM from serum-deprivation (2% FBS) or 10% FBS microenvironment (p < 0.05). Conclusion These results indicate that it is safe, effective, and lasting in effect to treat chronic wounds with SVF-gel and mechanisms of action that include secreting various cytokines and promoting cell proliferation and migration ability. Trial registration: Chinese Clinical Trail Registry, ChiCTR2000034624. Registered 12 July 2020—Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=5605

Variation in Stripe Rust Resistance and Morphological Traits in Wild Emmer Wheat Populations

Wild emmer wheat (Triticum dicoccoides), the tetraploid progenitor of cultivated wheats, is indigenous to the Near East Fertile Crescent. An important center of distribution is found today in and around the catchment area of the upper Jordan Valley in Israel and surrounding regions. In the current study, the field stripe rust resistance and morphological traits were analyzed using 98 sample accessions that represented the geographical distribution of wild emmer populations in Israel and its vicinity. The resistance tests at two field locations revealed that the majority of the wild emmer accessions possess quantitative resistance against stripe rust. This could be due to the high frequency of Yr36 in the wild emmer populations. The identification of potentially novel stripe rust resistance in this set of germplasm is highly significant. In total, 11 morphological traits were examined in this study. Wide range of natural variation was revealed in the tested morphological traits. Most of the morphological traits had significant correlations with climate variables, indicating that the local environmental conditions have a profound effect on shaping the genetic structure of wild emmer wheat. Our results suggest that wild emmer wheat has the enormous potential to improve stripe rust resistance and various important agronomical traits in wheat

Improved Early Postresuscitation EEG Activity for Animals Treated with Hypothermia Predicted 96 hr Neurological Outcome and Survival in a Rat Model of Cardiac Arrest

Purpose. To investigate the effect of hypothermia on 96 hr neurological outcome and survival by quantitatively characterizing early postresuscitation EEG in a rat model of cardiac arrest. Materials and Methods. In twenty male Sprague-Dawley rats, cardiac arrest was induced through high frequency transesophageal cardiac pacing. Cardiopulmonary resuscitation was initiated after 5 mins untreated arrest. Immediately after resuscitation, animals were randomized to either 2 hrs of hypothermia (N=10) or normothermia (N=10). EEG, ECG, aortic pressure, and core temperature were continuously recorded for 6 hrs. Neurological outcome was evaluated daily during the 96 hrs postresuscitation period. Results. No differences in the baseline measurements and resuscitation outcome were observed between groups. However, 96 hr neurological deficit score (204 ± 255 versus 500 ± 0, P=0.005) and survival (6/10 versus 0/10, P=0.011) were significantly better in the hypothermic group. Quantitative analysis of early postresuscitation EEG revealed that burst frequency and spectrum entropy were greatly improved in the hypothermic group and correlated with 96 hr neurological outcome and survival. Conclusion. The improved burst frequency during burst suppression period and preserved spectrum entropy after restoration of continuous background EEG activity for animals treated with hypothermia predicted favorable neurological outcome and survival in this rat model of cardiac arrest

Optimization on Preparation Conditions of Salidroside Liposome and Its Immunological Activity on PCV-2 in Mice

The aim of this study was to optimize the preparation conditions of salidroside liposome with high encapsulation efficiency (EE) and to study the immunological enhancement activity of salidroside liposome as porcine circovirus type 2 virus (PCV-2) vaccine adjuvant. Response surface methodology (RSM) was selected to optimize the conditions for the preparation of salidroside liposome using Design-Expert V8.0.6 software. Three kinds of salidroside liposome adjuvants were prepared to study their adjuvant activity. BALB/c mice were immunized with PCV-2 encapsulated in different kinds of salidroside liposome adjuvants. The PCV-2-specific IgG in immunized mice serum was determined with ELISA. The results showed that when the concentration of ammonium sulfate was 0.26 mol·L−1, ethanol volume 6.5 mL, temperature 43°C, ethanol injection rate 3 mL·min−1, and salidroside liposome could be prepared with high encapsulation efficiency of 94.527%. Salidroside liposome as adjuvant could rapidly induce the production of PCV-2-specific IgG and salidroside liposome I adjuvant proved to provide the best effect among the three kinds of salidroside liposome adjuvants

N-Methyl-d-aspartate (NMDA) Receptor NR2 Subunit Selectivity of a Series of Novel Piperazine-2,3-dicarboxylate Derivatives: Preferential Blockade of Extrasynaptic NMDA Receptors in the Rat Hippocampal CA3-CA1 Synapse

N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (−) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 μM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes

Upregulation of miR-196b-5p attenuates BCG uptake via targeting SOCS3 and activating STAT3 in macrophages from patients with long-term cigarette smoking-related active pulmonary tuberculosis

Abstract Background Cigarette smoking (CS) triggers an intense and harmful inflammatory response in lungs mediated by alveolar and blood macrophages, monocytes, and neutrophils and is closely associated with prevalence of tuberculosis (TB). The risk of death in patients with long-term cigarette smoking-related pulmonary tuberculosis (LCS-PTB) is approximately 4.5 times higher than those with nonsmoking pulmonary tuberculosis (N-PTB). However, the mechanisms underlying the harmful inflammatory responses in the setting of LCS-PTB have not been well documented. Methods 28 cases LCS-PTB patients, 22 cases N-PTB patients and 20 cases healthy volunteers were enrolled in this study. Monocytes were isolated from peripheral blood mononuclear cells. Differentiated human MDM and U937 cell were prepared with M-CSF and PMA stimulation, respectively. The miR-196b-5p, STAT1, STAT3, STAT4, STAT5A, STAT5B, STAT6, SOCS1 and SOCS3 mRNA expression were detected by qRT-PCR. Western blot was performed according to SOCS1, SOCS3, and pSTAT3 expression. The mycobacterial uptake by MDMs from different groups of patients after Bacillus Calmette–Guérin (BCG) infection and agomir-196b-5p or antagomir-196b-5p transfection were used by flow cytometry analysis. Human IL-6, IL-10 and TNF-α levels on the plasma and cell culture supernatant samples were measured using ELISA. For dual-luciferase reporter assay, the SOCS3 3′-UTR segments, containing the binding elements of miR-196b-5p or its mutant versions were synthesized as sense and antisense linkers. Results In this study, we found that IL-6, TNF-α production, SOCS3 mRNA expression were downregulated, while miR-196b-5p and STAT3 mRNA expression were upregulated in monocytes from LCS-PTB patients as compared to N-PTB patients. Meanwhile, we demonstrated that miR-196b-5p could target SOCS3 and activate STAT3 signaling pathway, which may possibly contribute to attenuation of BCG uptake and decrease in IL-6 and TNF-α production in macrophages. Conclusions Our findings revealed that CS exposure regulates inflammatory responses in monocyte/macrophages from LCS-PTB patients via upregulating miR-196b-5p, and further understanding of the specific role of miR-196b-5p in inflammatory responses mightfacilitate elucidating the pathogenesis of LCS-PTB, thus leading to the development of new therapeutic strategies for PTB patients with long-term cigarette smoking