Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.     

Serum 25-Hydroxyvitamin D Levels and Dry Eye Syndrome: Differential Effects of Vitamin D on Ocular Diseases

Purpose

To investigate associations between serum 25-hydroxyvitamin D levels and dry eye syndrome (DES), and to evaluate the differential effect of vitamin D on ocular diseases including age-related macular disease (AMD), diabetic retinopathy (DR), cataract, and DES.

Methods

A total of 16,396 participants aged >19 years were randomly selected from the Korean National Health and Nutrition Examination Survey. All participants participated in standardized interviews, blood 25-hydroxyvitamin D level evaluations, and comprehensive ophthalmic examinations. DES was defined by a history of clinical diagnosis of dry eyes by a physician. The association between vitamin D and DES was compared to the associations between vitamin D and AMD, DR, cataract, and DES from our previous studies.

Results

The odds of DES non-significantly decreased as the quintiles of serum 25-hydroxyvitamin D levels increased (quintile 5 versus 1, OR = 0.85, 95%CI: 0.55–1.30, P for trend = 0.076) after adjusting for potential confounders including age, sex, hypertension, diabetes, smoking status, and sunlight exposure times. The relative odds of DES (OR = 0.70, 95% CI: 0.30–1.64) and cataract (OR = 0.76, 95% CI: 0.59–0.99) were relatively high, while those of DR (OR = 0.37, 95% CI: 0.18–0.76) and late AMD (OR = 0.32, 95% CI: 0.12–0.81) were lower in men.

Conclusions

The present study does not support an association between serum 25-hydroxyvitamin D levels and DES. The preventive effect of serum 25-hydroxyvitamin D may be more effective for DR and late AMD than it is for cataract and DES.     

The Prevalence and Incidence of Atrial Fibrillation in Patients with Acute Pulmonary Embolism

Background

Symptomatic pulmonary embolism (PE) is a major cause of cardiovascular death and morbidity. Estimated prevalence and incidence of atrial fibrillation (AF) in developed countries are between 388–661 per 100,000, and 90–123 per 100,000 person-years respectively. However, the prevalence and incidence of AF in patients presenting with an acute PE and its predictors are not clear.

Methods

Individual patient clinical details were retrieved from a database containing all confirmed acute PE presentations to a tertiary institution from 2001–2012. Prevalence and incidence of AF was tracked from a population registry by systematically searching for AF during any hospital admission (2000–2013) based on International Classification of Disease (ICD-10) code.

Results

Of the 1,142 patients included in this study, 935 (81.9%) had no AF during index PE admission whilst 207 patients had documented baseline AF (prevalence rate 18,126 per 100,000; age-adjusted 4,672 per 100,000). Of the 935 patients without AF, 126 developed AF post-PE (incidence rate 2,778 per 100,000 person-years; age-adjusted 984 per 100,000 person-years). Mean time from PE to subsequent AF was 3.4 ± 2.9 years. Total mortality (mean follow-up 5.0 ± 3.7 years) was 42% (n = 478): 35% (n = 283), 59% (n = 119) and 60% (n = 76) in the no AF, baseline AF and subsequent AF cohorts respectively. Independent predictors for subsequent AF after acute PE include age (hazard ratio [HR] 1.06, 95% confidence interval [CI] 1.04–1.08, p<0.001), history of congestive cardiac failure (HR 1.88, 95% CI 1.12–3.16, p = 0.02), diabetes (HR 1.72, 95% CI 1.07–2.77, p = 0.02), obstructive sleep apnea (HR 4.83, 1.48–15.8, p = 0.009) and day-1 serum sodium level during index PE admission (HR 0.94, 95% CI 0.90–0.98, p = 0.002).

Conclusions

Patients presenting with acute PE have a markedly increased age-adjusted prevalence and subsequent incidence of AF. Screening for AF may be of importance post-PE.     

Upregulation of KLHDC4 Predicts a Poor Prognosis in Human Nasopharyngeal Carcinoma

Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification (P <0.05), N classification (P <0.05) and total staging (P <0.01) in NPC, and patients with higher KLHDC4 expression had poorer overall (P <0.01) and metastasis-free survival (P <0.05) rates. Knockout (KO) of KLHDC4 via CRISPR/Cas9-mediated gene editing in NPC cell line dramatically inhibited cell proliferation, colony formation in soft agar and tumor formation in nude mice. In addition, cell migration and invasion were also impaired by KLHDC4 depletion as revealed by wound healing and Transwell assay. Mechanically, loss of KLHDC4 markedly induced spontaneous apoptosis in NPC cells, as evidenced by increased levels of cleaved caspase-3 and cleaved PARP. Consistently, KLHDC4 knockout cell-derived xenografts also showed elevated cleaved caspase-3 and PARP but reduced Ki-67 staining. In conclusion, our results suggest that KLHDC4 promotes NPC oncogenesis by suppressing cellular apoptosis. Thus, KLHDC4 may serve as a prognosis biomarker and a potential therapeutic target for NPC.     

Steroidal Saponins from the Rhizomes of Aspidistra typica

Eleven new furostanol saponins, typaspidosides B-L (1–11), one new spirostanol saponin, typaspidoside M (12), and five known spirostanol saponins, 25S-atropuroside (13), neoaspidistrin (14), (25S)-pratioside D₁ (15), 25S-aspidistrin (16) and 25S-neosibiricoside (17) were isolated from the rhizomes of Aspidistra typica Baill. The structures of the new compounds were established using 1D and 2D NMR (¹H-¹H COSY, HMQC, HMBC and ROESY) spectroscopy, high resolution mass spectrometry, and chemical methods. The aglycones of 1–3 (unusual furostanol saponins with opened E ring type), 9 and 10 (the methoxyl substituent at C-23 position) were found, identified from natural products for the first time. Moreover, the anti-HIV activities of the isolated steroidal glycosides were assessed, and compounds 13, 14, 16 and 17 exhibited high active against HIV-1.     

Inhibition of TREM-1 and Dectin-1 Alleviates the Severity of Fungal Keratitis by Modulating Innate Immune Responses

Purpose

To explore the possibility that inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) and Dendritic cell-associated C-type lectin-1(Dectin-1) could modulate the innate immune response and alleviate the severity of corneal fungal keratitis.

Method

TREM-1 and Dectin-1 expression was detected in fungus-infected human corneal specimens by real-time PCR. C57BL/6 (B6) mice were injected with Aspergillus fumigatus and divided into 4 groups that received subconjunctival injections of PBS and IgG as a control (group I), mTREM-1/IgG fusion protein (group II), the soluble β-glucan antagonist laminarin (group III), or mTREM-1/Fc and laminarin (group IV). Corneal virulence was evaluated based on clinical scores. TREM-1 and Dectin-1 mRNA levels were assayed using real-time PCR. The distribution patterns of TREM-1, Dectin-1 and cellular infiltrates in fungus-infected corneas were examined by immunohistochemistry. Moreover, changes in T Helper Type1 (Th1)-/ T Helper Type1 (Th2)- type cytokines and proinflammatory cytokines were measured.

Results

The expression of TREM-1 and Dectin-1 increased significantly and correlated positively with the progression of fungal keratitis. Most infiltrated cells were neutrophils and secondarily macrophages in infected cornea. The clinical scores decreased after interfering with TREM-1 and Dectin-1 expression in infected mouse corneas. Levels of Th1-type cytokines including interleukin-12 (IL-12), IL-18 and interferon-γ (IFN-γ) were decreased in the cornea, while the levels of Th2-type cytokines, including IL-4, IL-5 and IL-10, showed obvious increases.

Conclusion

TREM-1 and Dectin-1 function concurrently in the corneal innate immune response by regulating inflammatory cytokine expression in fungal keratitis. Inhibition of TREM-1 and Dectin-1 can alleviate the severity of corneal damage by downregulating the excessive inflammatory response.     

Geriatric Nutritional Risk Index (GNRI) Independently Predicts Amputation Inchronic Criticallimb Ischemia (CLI)

Objective

General malnutrition usually occurs in critical limb ischemia (CLI) patients because of shortness of appetite and sleeplessness leaded by chronic pain. And amputation frequently is end-point of CLI patients. So the aim of this study was to assess the predictive ability of Geriatric nutritional risk index (GNRI) for predicting amputation in patients with CLI.

Methods

A retrospective study was designed. Demographics, history, comorbidity, and risk factors for peripheral vascular disease of admitted patients, and laboratory study were documented. Patients’ height, weight and BMI were recorded. Amputation was identified as end-point during follow-up. Patients’ amputation-free survival (AFS) was recorded.

Result

172 patients were identified, with mean age 71.98±3.12. Geriatric nutritional risk index (GNRI) = 90 was taken as cutoff value of high risk of amputation for CLI patients via using receiver operating characteristic (ROC) curve. Span of follow-up was 12–48 months. During follow-up, 60 patients (36.04%) received amputation surgery. And analyzed by Cox proportional hazards model, it is found that GNRI was the independent predictive factor for amputation in long term.

Conclusion

This study revealed that GNRI was a reliable and effective predictive marker for AFS. GNRI could identify patients with high risk for amputation in early time.     

Expression of VSTM1-v2 Is Increased in Peripheral Blood Mononuclear Cells from Patients with Rheumatoid Arthritis and Is Correlated with Disease Activity

Rheumatoid arthritis (RA) is a chronic, systematic autoimmune disease that mainly affects joints and bones. Although the precise etiology is still unknown, Th17 cell is being recognized as an important mediator in pathogenesis of RA. VSTM1-v2 is a novel cytokine which has recently been reported to promote the differentiation of Th17 cells. This study is performed to study whether VSTM1-v2 can be recognized as a biomarker of RA, and is correlated to IL-17 expression. We obtained peripheral blood mononuclear cells (PBMCs) from 40 patients with RA and 40 age- and sex- matched healthy controls by standard Ficoll-Paque Plus density centrifugation. The mRNA expression levels of VSTM1-v2 and IL-17A in PBMCs were detected by real time-PCR. Disease activity parameters of RA were measured by routine methods. Our results showed that VSTM1-v2 mRNA expression in PBMCs from RA patients was significantly increased in comparison of that in healthy individuals. The VSTM1-v2 mRNA expression level was positively correlated with IL-17A mRNA expression level, DAS28, CRP and ESR, but was not correlated to RF, Anti-CCP or ANA. VSTM1-v2 might be a biomarker of RA and a novel factor in the pathogenesis of RA.     

MiR-126 Suppresses the Glucose-Stimulated Proliferation via IRS-2 in INS-1 β Cells

Background

Increasing evidence suggests that miR-126 participates in the glucose homeostasis through its target molecules. Although bioinformatics analysis predicts that miR-126 can bind with the insulin receptor substrate-2(IRS-2) mRNA at the “seed sequence”, but there are still no definitely reports to support it. In this study, we provided evidences that IRS-2 was one of the target genes of miR-126. And miR-126 has a proliferation inhibiting effects in INS-1 β cells, mainly through the suppression of IRS-2.

Methods

The 3’-UTR of IRS-2 regulated by miR-126 was analyzed by the luciferase assay and western blot. Furthermore, proliferation of INS-1 β cells stimulated by glucose was tested, and the association between IRS-2 and miR-126 were analyzed.

Results

We found that mutation of only three of the 6 “seed sequences” can eliminate the inhibition effect of miR-126. In INS-1 β cells, administration of miR-126 suppresses the proliferation, together with the unbalanced down-regulation of IRS-2 and IRS-1. Over-expression of IRS-2 can reverse the proliferation effect of miR-126, while not of IRS-1. These results suggested that miR-126 inhibited the β-cell proliferation via the inhibition of IRS-2 instead of IRS-1.Additionally, we also found that high glucose and insulin could stimulate the rapid production of endogenous miR-126 within 6 hours, together with the short term suppression of IRS-1 and IRS-2 expression, and intensify the unbalanced expression of IRS-1 and IRS-2.

Conclusions

IRS-2 was one of the targets of miR-126. MiR-126 inhibited the β-cell proliferation through IRS-2 instead of IRS-1. MiR-126 may take part in the glucose homeostasis both through its target IRS-2 and IRS-1. The unbalance between IRS-1 and IRS-2 caused by miR-126 may play an important role in type 2 diabetes.     

Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016