A cellular platform for production of C4 monomers.

Living organisms carry out a wide range of remarkable functions, including the synthesis of thousands of simple and complex chemical structures for cellular growth and maintenance. The manipulation of this reaction network has allowed for the genetic engineering of cells for targeted chemical synthesis, but it remains challenging to alter the program underlying their fundamental chemical behavior. By taking advantage of the unique ability of living systems to use evolution to find solutions to complex problems, we have achieved yields of up to ∼95% for three C4 commodity chemicals, n-butanol, 1,3-butanediol, and 4-hydroxy-2-butanone. Genomic sequencing of the evolved strains identified pcnB and rpoBC as two gene loci that are able to alter carbon flow by remodeling the transcriptional landscape of the cell, highlighting the potential of synthetic pathways as a tool to identify metabolic control points.

Erythroferrone contributes to iron mobilization for embryo erythropoiesis in iron-deficient mouse pregnancies.

Erythroferrone (ERFE) is an erythroblast-secreted regulator of iron metabolism. The production of ERFE increases during stress erythropoiesis, leading to decreased hepcidin expression and mobilization of iron. Pregnancy requires a substantial increase in iron availability to sustain maternal erythropoietic expansion and fetal development and is commonly affected by iron deficiency. To define the role of ERFE during iron-replete or iron-deficient pregnancy, we utilized mouse models expressing a range of ERFE levels: transgenic (TG) mice overexpressing ERFE, wild-type (WT), and ERFE knockout (KO) mice. We altered maternal iron status using diets with low or standard iron content and performed the analysis at E18.5. Iron deficiency increased maternal ERFE in WT pregnancy. Comparing different maternal genotypes, ERFE TG dams had lower hepcidin relative to their liver iron load but similar hematological parameters to WT dams on either diet. In ERFE KO dams, most hematologic and iron parameters were comparable to WT, but mean corpuscular volume (MCV) was decreased under both iron conditions. Similar to dams, TG embryos had lower hepcidin on both diets, but their hematologic parameters did not differ from those of WT embryos. ERFE KO embryos had lower MCV than WT embryos on both diets. The effect was exacerbated under iron-deficient conditions where ERFE KO embryos had higher hepcidin, lower Hb and Hct, and lower brain iron concentration compared to WT embryos, indicative of iron restriction. Thus, under iron-deficient conditions, maternal and embryo ERFE facilitate iron mobilization for embryonic erythropoiesis.

Distinct transcriptomic response to Newcastle disease virus infection during heat stress in chicken tracheal epithelial tissue.

Newcastle disease (ND) has a great impact on poultry health and welfare with its most virulent (velogenic) strain. In addition, issues exacerbated by the increase in global temperatures necessitates a greater understanding of the host immune response when facing a combination of biotic and abiotic stress factors in poultry production. Previous investigations have revealed that the host immune response is tissue-specific. The goal of this study was to identify genes and/or signaling pathways associated with immune response to NDV (Newcastle disease virus) in the trachea, an essential organ where NDV replicate after the infection, by profiling the tissue specific transcriptome response in two genetically distinct inbred chicken lines when exposed to both abiotic and biotic stressors. Fayoumis appear to be able to respond more effectively (lower viral titer, higher antibody levels, immune gene up-regulation) and earlier than Leghorns. Our results suggest NDV infection in Fayoumis appears to elicit proinflammatory processes, and pathways such as the inhibition of cell viability, cell proliferation of lymphocytes, and transactivation of RNA, more rapidly than in Leghorns. These differences in immune response converge at later timepoints which may indicate that Leghorns eventually regulate its immune response to infection. The profiling of the gene expression response in the trachea adds to our understanding of the chicken host response to NDV infection and heat stress on a whole genome level and provides potential candidate genes and signaling pathways for further investigation into the characterization of the time-specific and pathway specific responses in Fayoumis and Leghorns.

Functional responses between PMP3 small membrane proteins and membrane potential.

The Plasma Membrane Proteolipid 3 (PMP3, UPF0057 family in Uniprot) family consists of abundant small hydrophobic polypeptides with two predicted transmembrane helices. Plant homologues were upregulated in response to drought/salt-stresses and yeast deletion mutants exhibited conditional growth defects. We report here abundant expression of Group I PMP3 homologues (PMP3(i)hs) during normal vegetative growth in both prokaryotic and eukaryotic cells, at a level comparable to housekeeping genes, implicating the regular cellular functions. Expression of eukaryotic PMP3(i)hs was dramatically upregulated in response to membrane potential (Vm) variability (Vmvar ), whereas PMP3(i)hs deletion-knockdown led to Vm changes with conditional growth defects. Bacterial PMP3(i)h yqaE deletion led to a shift of salt sensitivity; Vmvar alternations with exogenous K+ addition downregulated prokaryotic PMP3(i)hs, suggesting [K+ ]-Vmvar axis being a significant feedback element in prokaryotic ionic homeostasis. Remarkably, the eukaryotic homologues functionally suppressed the conditional growth defects in bacterial deletion mutant, demonstrating the conserved cross-kingdom membrane functions by PMP3(i)hs. These data demonstrated a direct reciprocal relationship between PMP3(i)hs expression and Vm differentials in both prokaryotic and eukaryotic cells. Cumulative with PMP3(i)hs ubiquitous abundance, their lipid-binding selectivity and membrane protein colocalization, we propose [PMP3(i)hs]-Vmvar axis as a key element in membrane homeostasis.

Perceptions of risk and reward in BRCA1 and BRCA2 mutation carriers choosing salpingectomy for ovarian cancer prevention.

Salpingectomy with interval oophorectomy has gained traction as an ovarian cancer prevention strategy, but is not currently recommended for high risk women. Nevertheless, some choose this approach. We aimed to understand risk perception and plans for oophorectomy in BRCA1 and BRCA2 (BRCA) mutation carriers choosing salpingectomy for ovarian cancer prevention. This was a longitudinal survey study of BRCA mutation carriers who underwent bilateral salpingectomy to reduce ovarian cancer risk. An initial written questionnaire and telephone interview was followed by annual phone interviews. 22 women with BRCA mutations were enrolled. Median follow-up was three years. The median age at salpingectomy was 39.5 years (range 27-49). Perceived lifetime ovarian cancer risk decreased by half after salpingectomy (median risk reduction 25%, range 0-40%). At final follow-up, five (22.7%) had undergone oophorectomy and five women (22.7%) were not planning to undergo completion oophorectomy. BRCA mutation carriers who had salpingectomy after the recommended age of prophylactic surgery (vs. before the recommended age) were less likely to plan for future oophorectomy (28.6% vs. 66.7%, p = 0.037). All women were satisfied with their decision to undergo salpingectomy with eighteen (81.8%) expressing decreased cancer-related worry. There were no diagnoses of ovarian cancer during our study period. In conclusion, most BRCA mutation carriers undergoing risk-reducing salpingectomy are satisfied with their decision and have lower risk perception after salpingectomy, though some older mutation carriers did not plan on future oophorectomy. Salpingectomy with delayed oophorectomy in BRCA mutation carriers remains investigational and should preferably be performed within a clinical trial to prevent introduction of an innovation before safety has been proven.

Integrated Proteomic and Transcriptomic Analysis of Differential Expression of Chicken Lung Tissue in Response to NDV Infection during Heat Stress.

: Newcastle disease virus (NDV) is a devastating worldwide poultry pathogen with major implications for global food security. In this study, two highly inbred and genetically distinct chicken lines, Fayoumis and Leghorns, were exposed to a lentogenic strain of NDV, while under the effects of heat stress, in order to understand the genetic mechanisms of resistance during high ambient temperatures. Fayoumis, which are relatively more resistant to pathogens than Leghorns, had larger numbers of differentially expressed genes (DEGs) during the early stages of infection when compared to Leghorns and subsequently down-regulated their immune response at the latter stages to return to homeostasis. Leghorns had very few DEGs across all observed time points, with the majority of DEGs involved with metabolic and glucose-related functions. Proteomic analysis corroborates findings made within Leghorns, while also identifying interesting candidate genes missed by expression profiling. Poor correlation between changes observed in the proteomic and transcriptomic datasets highlights the potential importance of integrative approaches to understand the mechanisms of disease response. Overall, this study provides novel insights into global protein and expression profiles of these two genetic lines, and provides potential genetic targets involved with NDV resistance during heat stress in poultry.

Noncytotoxic-Related Primary Ovarian Insufficiency in Adolescents: Multicenter Case Series and Review.

STUDY OBJECTIVE: Primary ovarian insufficiency (POI) in adolescents not due to cytotoxic therapy has not been well studied. Causes of POI have been described in adults, but adolescents might represent a unique subset necessitating a targeted approach to diagnosis, workup, and treatment. We sought to better characterize adolescent POI through a descriptive multicenter study. DESIGN: Case series of patients with POI. SETTING: Six tertiary care institutions. PARTICIPANTS: Patients presenting from 2007 to 2014 aged 13-21 years diagnosed with noncytotoxic POI, with exclusions for those who received gonadotoxic therapy, with 46XY gonadal dysgenesis, or lack of evidence of hypergonadotropic hypogonadism on chart review. INTERVENTIONS: Review and data extraction of records identified according to International Classification of Diseases Ninth or Tenth Revision codes. MAIN OUTCOME MEASURES: Data were analyzed for signs and symptoms, workup, and treatments. Complete workup was on the basis of American College of Obstetricians and Gynecologists guidelines. Characteristics of patients with POI who presented with delayed puberty/primary amenorrhea vs secondary amenorrhea were compared. RESULTS: One hundred thirty-five records were identified. Those who had received cytotoxic therapy (n = 52), 46XY gonadal dysgenesis (n = 7), or on review did not have POI (n = 19) were excluded. Of 57 remaining cases, 16 were 45X, 2 had galactosemia, and 4 had X-chromosome abnormalities. Most did not undergo full etiologic evaluation. Girls diagnosed after primary amenorrhea/delayed puberty were less symptomatic and more likely to receive an estrogen patch than those diagnosed after secondary amenorrhea. CONCLUSION: Noncytotoxic POI in adolescents is an uncommon condition with, to our knowledge, only 64 cases in 6 institutions over 7 years. These patients might not undergo complete etiological workup. Aside from 45X, the most common etiologies were X-chromosome abnormalities or galactosemia.

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae.

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

BACKGROUND: Economical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. Neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. In addition to cellulases and hemicellulases, N. crassa secretes lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose by generating oxidized sugars-particularly aldonic acids. However, the strategies N. crassa employs to utilize these sugars are unknown. RESULTS: We identified an aldonic acid utilization pathway in N. crassa, comprised of an extracellular hydrolase (NCU08755), cellobionic acid transporter (CBT-1, NCU05853) and cellobionic acid phosphorylase (CAP, NCU09425). Extracellular cellobionic acid could be imported directly by CBT-1 or cleaved to gluconic acid and glucose by a ß-glucosidase (NCU08755) outside the cells. Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP. However, it remains unclear how N. crassa utilizes extracellular gluconic acid. The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions. CONCLUSIONS: We successfully identified a branched aldonic acid utilization pathway in N. crassa and transferred its essential components into S. cerevisiae, a robust industrial microorganism.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

Anti-proliferative activities of sinigrin on carcinogen-induced hepatotoxicity in rats.

Liver cancer is one of the leading causes of cancer death worldwide. A very high incidence of new liver cancer cases is diagnosed every year, and metastasis has been found to correlate to poor prognoses in humans. Better treatments for liver cancer are thus clearly needed. Sinigrin is one of the major ingredients present in Brassica nigra, which has been used in combination with other herbs for treatment of various diseases. The anti-proliferative activities of sinigrin were studied in a model of carcinogen-induced hepatotoxicity in rats. Rats were orally administered with sinigrin on a daily basis for three months before sacrifice. Sinigrin was found to significantly inhibit the proliferation of liver tumor cells; the number of surface tumors in the rat liver was dramatically reduced. Sinigrin induced apoptosis of liver cancer cells through up-regulation of p53 and down-regulation of Bcl-2 family members and caspases. Our findings indicated that the liver functions were gradually restored after treatment with sinigrin and that the agent did not cause liver toxicity. Cell cycle analysis indicated that sinigrin caused cell cycle arrest in G0/G1 phase. The results suggest that sinigrin exerts important anti-proliferative activities in carcinogen-induced hepatocarcinogenesis in rats, and highlight the potential of sinigrin as an anti-cancer agent for liver cancer.

Tetrandrine inhibits hepatocellular carcinoma cell growth through the caspase pathway and G2/M phase.

Activation of p53-independent pathways plays an important role in phytochemical-induced apoptosis and is considered to be a crucial factor in the invasion and metastasis of cancer. Previous studies have shown that combined effects of Stephania tetrandra with medicinal herbs exhibit beneficial effects in cancer patients. Tetrandrine, an active component of Stephania tetrandra has been reported to have anticancer properties in cancer cells. However, the mechanism(s) of action of tetrandrine in liver cancer have yet to be fully elucidated. In this study, we investigated the effects of tetrandrine in hepatocellular carcinoma (HCC) cells. The results showed that tetrandrine inhibited HCC cell proliferation by suppression of cell cycle progression at the G2/M phase. Changes in the expression levels of Bax, Bcl, p53, survivin, PCNA, PARP and p21 were observed. In addition, tetrandrine increased caspase-3 expression and induced DNA fragmentation in Huh-7 cells. The results suggest that the anti-cancer effect of tetrandrine in Huh-7 cells may be mediated by p53-independent pathways.

Balloon catheter dilatation of Eustachian tube: a preliminary study.

OBJECTIVE: Eustachian tube dysfunction is a common problem and transnasal endoscopic balloon dilation of the Eustachian tube (ET) is a new surgical technique. The goal of this study is to review the evolution of this novel technique and study the preliminary outcomes. SUBJECTS AND METHODS: Balloon catheter dilation of the 100 Eustachian tubes in 70 adults was performed at a tertiary medical center from January 2009 to January 2011. A 5-mm sinus balloon catheter was endoscopically placed transnasally into the proximal ET to dilate the cartilaginous ET. Cases were reviewed with respect to indications, outcomes, and complications. RESULTS: Of the 100 ETs, ear fullness and pressure were improved in 71% of patients studied for 26.3 weeks (± 3.6). Of 8 patients followed for a minimum of 34 months, 87% reported persistent improvement. One complication is reported. CONCLUSION: Endoscopic transnasal ET balloon dilation is a novel approach to treating ET dysfunction. Benefits can be durable up to 3 years. This technique holds much promise and merits further investigation.

Active phytochemicals from Chinese herbs as therapeutic agents for the heart.

Naturally occurring plant alkaloids, in particular those identified from herbal medicines, are finding therapeutic use. Heart diseases can be well managed with specific formulations of herbal medicines. The combined action of multiple constituents of herbal medicines works with therapeutic benefits in humans. The established formulations of Traditional Chinese medicines show efficacy in treatment of diseases. However, individual herbal principles seldom show pharmacological activity. Nevertheless, some of the active alkaloids and terpenoids from medicinal herbs have been identified. The pharmacological activities of these herbal compounds have been studied. These active constituents of herbal medicine are also used in nutrient supplements, but the modes of action of the active component remain sketchy. The present review describes the recent development of those active principles from herbal medicines as cardiovascular agents. The study will provide insights into herbal medicines for drug development for the treatment of cardiovascular disease.

Recombinant expression of His-tagged saposin B and pH-dependent binding to the lipid coenzyme Q10.

The use of coenzyme Q10 (CoQ10) has been increasing rapidly during recent years due to its postulated beneficial properties in human health, providing energy and antioxidant protection. There are no known negative side effects of CoQ10 even at very high levels. Recently, native saposin B (sapB) has been shown to bind CoQ10 and subsequently be excreted. It is thought that this interaction between sapB and CoQ10 could be a mechanism to avoid any possible CoQ10 toxicity. The interaction between sapB and CoQ10 is poorly understood. Here we present an increased fermentative yield of recombinant sapB and demonstrate that recombinant sapB will bind CoQ10 in a pH-dependent manner similar to sapB binding with other lipids. SapB was coated onto an IMAC (immobilized metal affinity chromatography) resin and successfully bound CoQ10 at pH 5.0 with release of the CoQ10 at pH 9.0.

Effect of sialodacryoadenitis virus infection on axonal regeneration.

The effect of sialodacryoadenitis virus (SDAV) infection on axonal regeneration and functional recovery was investigated in male Lewis rats. Animals underwent unilateral tibial nerve transection, immediate repair, and treatment with either FK506 (treated) or control vehicle (untreated). Serial walking track analyses were performed to assess functional recovery. Nerves were harvested for morphometric analysis on postoperative day 18 after an SDAV outbreak occurred that affected the 12 experimental animals. Histomorphometry and walking track data were compared against 36 historical controls. Rats infected with SDAV demonstrated severely impaired axonal regeneration and diminished functional recovery. Total fiber counts, nerve density, and percent neural tissue were all significantly reduced in infected animals (P < 0.05). Active SDAV infection severely impaired nerve regeneration and negated the positive effect of FK506 on nerve regeneration in rats. Immunosuppressive risks must be weighed carefully against the potential neuroregenerative benefits in the treatment of peripheral nerve injuries.

A feasibility study to evaluate a novel drug delivery technique through nasal/sinus mucosa using a biodegradable polymer in a guinea pig model.

OBJECTIVE: Targeted topical pharmaceuticals have fewer side effects than systemic therapy and present an interesting option to treat chronic sinus disease. A simple, dependable, resorbable drug delivery mechanism has been elusive. The goal of this study is to examine the feasibility of a novel bioresorbable synthetic polymer for drug delivery in nose and sinuses. STUDY DESIGN: Feasibility study. SETTING: Animal study. SUBJECTS AND METHODS: Polyurethane sponges soaked in either triamcinolone or gentamicin were placed in the ethmoid cavities of 14 guinea pigs via an external approach; 2 additional animals served as controls. Serum levels of each drug were assayed at intervals up to 21 days. Histopathological examination of the relevant sinonasal anatomy of each animal was performed after 21 days. RESULTS: Serum levels of each drug were detectable between days 1 and 21. There were no significant differences in the histopathological examination of nasal mucosa in guinea pigs in which either drug was applied compared with control animals in which the bioabsorbable material was soaked in saline. The polyurethane sponge did not cause any foreign body reaction, granuloma, or polypoidal change to the sinus mucosa. Two animals developed a subclinical infection at the surgical site. CONCLUSION: The targeted use of topical pharmaceuticals via a synthetic bioresorbable nasal sponge dressing in this guinea pig model demonstrated minimal systemic absorption and minimal histopathological changes. This technology is currently under investigation in human clinical trials.

Interstitial delivery of vascular endothelial growth factor to skin flaps.

OBJECTIVES: To demonstrate the feasibility of using microporous catheters to deliver a growth factor in a skin flap model, and to determine whether removal of excess fluid by ultrafiltration catheters reduces edema. METHODS: In a controlled study at a research laboratory associated with major teaching hospital, vascular endothelial growth factor was delivered to porcine skin flaps by direct infusion using hollow fiber catheters. Treated flaps received either infusion alone or infusion and ultrafiltration via hollow fibers inserted into the distal portion of the flap. Controls had neither type of catheter placed. The main outcome measure was flap survival and edema. RESULTS: Treated anterior flaps were found to have increased survival (mean [SD] increase, 49.9% [9.4%]) compared with control flaps (44.1% [4.5%]) for group (P = .005) and side (P = .01) but not by interaction (P = .14). Water content was significant by analysis of variance for group, position, and interaction (all P < .001, df = 31) for treated (55.3% [9.7%]) and control (61.9% [8.2%]) groups. CONCLUSIONS: This study demonstrated feasibility of using hollow fiber technology to deliver a growth factor to skin flaps. Further study may yield clinical applications for human patients undergoing reconstructive procedures.