Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk‐based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 292–333, 2013.     

Collision/Reaction Cell ICP-MS with Shielded Torch and Sector Field ICP-MS for the Simultaneous Determination of Selenium Isotopes in Biological Matrices

The determinations of selenium isotopes in biological samples were performed using both inductively coupled plasma collision/reaction cell quadruple mass spectrometer (CRC-ICP-QMS) and inductively coupled plasma sector field mass spectrometers (SF-ICP-MS). To significantly decrease the argon-based interferences at m/z 74 (³⁶Ar³⁸Ar), 76 (³⁸Ar³⁸Ar, ⁴⁰Ar³⁶Ar), 78 (³⁸Ar⁴⁰Ar), and 80 (⁴⁰Ar⁴⁰Ar), the gas-flow rates of a helium and hydrogen mixture used in the collision cell were optimized to 1.0 mL/min H₂ and 3.5 mL/min He. Under the optimized condition, the precisions for natural selenium isotope ratio measurements of both instruments were evaluated and compared using 100 ppb Se standard solution. A modified external calibration quantification method was applied for the simultaneous determination of clinically used enriched selinocompounds (⁷⁷Se-selenate, ⁸²Se-selenite, ⁷⁶Se-methylseleninic acid^IV, ⁷⁸Se-methylselenonic acid^VI) and to examine their fate in rat organs (liver, kidney, and lung).     

Association of morphological and biochemical features of mulberry lines with resistance to bacterial leaf spot

Xanthomonas campestris pv. mori causes bacterial leaf spot (BLS) of mulberry (Morus spp.) worldwide. Twenty mulberry lines with contrasting response to BLS showed significant (p?<?0.01) variation between resistant and susceptible groups for stomata frequency (SF), trichome density (TD), total soluble sugar (TSS) and polyphenol (PP) content, while non-significant for chlorophyll content. Heritability (broad-sense) estimates obtained from the variance component analysis were high (≥0.72) for these traits. The disease severity index values of the lines were significantly negatively correlated with TD (r?=??0.66; p?<?0.01), TSS (r?=??0.73; p?<?0.01) and PP (r?=??0.61; p?<?0.01), while positively correlated with SF (r?=?0.65; p?<?0.01). The cluster analysis was based on highly significant four variables and DSI values, and classified the lines into two major clusters. The resistant lines that come under cluster-I had high TD, TSS and PP, while those in susceptible category had high SF in cluster-II. These findings suggest that direct selection for SF, TD, TSS and PP will be effective for development of host resistance against BLS in mulberry.     

Carboxy group derivatization for enhanced electron‐transfer dissociation mass spectrometric analysis of phosphopeptides

A novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. By tagging the carboxy group with 1‐(2‐pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron‐transfer dissociation MS. Besides, after PP‐derivatization, most non‐specific bindings can be avoided by eliminating the interaction between the carboxy group and TiO₂, greatly improving the specificity of TiO₂‐based phosphopeptide enrichment strategy. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC‐based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP‐derivatization method is promising for high‐throughput phosphoproteome research.     

Structural transitions in mixed ternary noble gas clusters

The properties of noble gas systems can be greatly extended by heterogeneous mixtures of elements. The geometrical structures and energies of mixed Ar–Kr–Xe clusters were investigated using ternary Lennard-Jones (TLJ) potential. For the Ar₁₉Kr _n Xe₁₉, Ar₁₉Kr₁₉Xe _n , and Ar _n Kr₁₉Xe₁₉ (n?=?0–17) clusters investigated, the results show that only two minimum energy configurations exist, i.e., polytetrahedron and six-fold pancake. The inner core of all these clusters is composed mainly of Ar atoms, and Kr and Xe atoms are distributed on the surface with well mixed pattern for polytetrahedral and segregate pattern for six-fold pancake configurations. The relative stability property of Ar–Kr–Xe clusters with a certain composition is discussed. Moreover, the role of heterogeneity on the strain was investigated, and reduced strain energies in Ar–Kr–Xe clusters were studied to find possible ways of reducing strain. The results showed that the strain energies were affected mainly by Ar–Ar, Ar–Kr, and Xe–Xe bonds.

Thermo-responsive non-woven scaffolds for "smart" 3D cell culture

The thermo‐responsive polymer poly(N‐isopropylacrylamide) has received widespread attention for its in vitro application in the non‐invasive, non‐destructive release of adherent cells on two dimensional surfaces. In this study, 3D non‐woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET), and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation were estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non‐woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available Algimatrix? alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non‐woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP‐g‐PNIPAAm‐A and PP‐g‐PNIPAAm‐B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non‐woven, thermo‐responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation. Biotechnol. Bioeng. 2012; 109:2147–2158. © 2012 Wiley Periodicals, Inc.     

Preparation and Characterization of a Silk Fibroin/Polyurethane Fiber Blend Membrane Containing Actinomycin X2 with Excellent Mechanical Properties and Enhanced Antibacterial Activities

Development of suitable antimicrobial biomaterials for hygienic wound dressing and healing is an important requirement for medical application. Durable mechanical properties increase the application range of biomaterial in different environmental and biological conditions. Due to the inherent brittleness of silk fibroin (SF), polyurethane fiber (PUF) was used to modify SF containing actinomycin X2 (Ac.X2) to prepare silk fibroin@actinomycin X₂/polyurethane fiber (ASF/PUF) blend membranes. The ASF/PUF blend membrane was developed by solution casting method. Incorporation of PUF improved the flexibility of material and introduction of Ac.X2 has increased antibacterial activity of materials. Excellent mechanical properties (tensile strength up to 25.7 MPa and elongation at break up to 946.5 %) of 50 % SF+50 % PUF blend membrane were proved by tensile testing machine. FT-IR spectra, TGA, contact angle and DMA were tested to prove the blend membrane's physico-chemical characteristics. ASF/PUF blend membrane displayed satisfactory antibacterial activity against S. aureus, and the cytotoxicity tests showed that the blend membrane has better biosafety compared to directly applied Ac.X2 in soluble form. These results suggest that the modification of SF through PUF for development of flexible antibacterial membranes has great potential application value in the field of silk-like material fabrication.     

Experimental and theoretical studies of the optical and electrical characteristics of a high-pressure pulsed discharge in argon

The optical and electrical characteristics of pulsed discharges in pure Ar at pressures of up to 7 atm, at which the discharge becomes unstable, are studied in a simple experimental device with automatic preionization. The gas temperature in the discharge is estimated from the width of the recorded emission spectrum. An analytical model of the vibrational relaxation of Ar ^*₂ (v) is used to better determine the constants of the vibrational-translational relaxation of Ar ^*₂ (v) molecules in their collisions with Ar atoms. The zerodimensional numerical model of a pulsed discharge in Ar is modified. The experimental and calculated results are compared in detail. Good agreement is achieved between the measured and calculated time dependences of the electrode voltage and the intensity of spontaneous emission in the pressure range of 1–6 atm, as well as between the measured and calculated values of the gas temperature at pressures of 3–6 atm. Preliminary results from numerical studies of the possibility of achieving generation are discussed.

     

Mulberry Paper‐Based Supercapacitor Exhibiting High Mechanical and Chemical Toughness for Large‐Scale Energy Storage Applications

In response to the demand for flexible and sustainable energy storage devices that exhibit high electrochemical performance, a supercapacitor system is fabricated using mulberry tree‐derived paper as a substrate and Poly(3,4‐ethylenedioxythiophene)‐poly(styrenesulfonate) (PEDOT:PSS) and carbon black as the active material. The mulberry paper‐based supercapacitor system demonstrates high energy density of 29.8–39.8 Wh kg^?1 and power density of 2.8–13.9 kW kg^?1 with 90.7% retention of its initial capacity over 15 000 charge–discharge cycles. In addition, the mulberry tree fibers are known to have superior mechanical strength and toughness and the mulberry paper‐based supercapacitor; as a result, exhibit high mechanical and chemical toughness; 99% of its initial capacity is retained after 100 repeated applications of bending strains, and twisting. 94% capacity retention is observed even after exposure to HCl and H₂SO₄ acid solutions. The fabrication methodology of the mulberry‐based supercapacitor is highly scalable and could be stacked to increase the energy storage capacity, where operation of light‐emitting diode lights with a drive voltage of 12 V integrated in a wearable device is demonstrated.     

Photoanalogues of the Initiation Substrates of the RNA Polymerase II, 5‐Azido‐2‐Nitrobenzoyl Derivatives of the ATP γ‐Amidophosphate: The Possible Photoinduced Degradation of the Functional Group to an N‐Arylhydroxylamine

Photoanlogues of the initiation substrates of the RNA polymerase II, N₃Ar‐ NH(CH₂)_nNHpppA where N₃Ar is 5‐azido‐2‐nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p‐nitro‐substituted aromatic azide in aqueous medium was investigated. Using the azoxy‐coupling reaction it was possible to determine whether a nitrene or p‐nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution.     

Plasma-treated silk fibroin nanofibers for skin regeneration

Silk fibroin (SF) nanofibers were prepared by electrospinning and treated with plasma in the presence of oxygen or methane gas to modify their surface characteristics. The surface characteristics of the SF nanofibers after plasma treatment were examined using contact angle measurements and XPS analysis. The hydrophilicity of the electrospun SF nanofibers decreased slightly by the CH₄ plasma treatment. On the other hand, the hydrophilicity of the SF nanofibers increased greatly by an O₂ plasma treatment. The O₂-treated SF nanofibers showed higher cellular activities for both normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) than the untreated ones.     

Balancing High Open Circuit Voltage over 1.0 V and High Short Circuit Current in Benzodithiophene‐Based Polymer Solar Cells with Low Energy Loss: A Synergistic Effect of Fluorination and Alkylthiolation

Based on the most recently significant progress within the last one year in organic photovoltaic research from either alkylthiolation or fluorination on benzo[1,2‐b:4,5‐b′]dithiophene moiety for high efficiency polymer solar cells (PSCs), two novel simultaneously fluorinated and alkylthiolated benzo[1,2‐b:4,5‐b′] dithiophene (BDT)‐based donor–acceptor (D–A) polymers, poly(4,8‐bis(5′‐((2″‐ethylhexyl)thio)‐4′‐fluorothiophen‐2′‐yl)benzo[1,2‐b:4,5‐b′]dithiophene‐2,6‐diyl)‐alt‐2′‐ethylhexyl‐3‐fluorothieno[3,4‐b]thiophene‐2‐carboxylate (PBDTT‐SF‐TT) and poly(4,8‐bis(5′‐((2″‐ethylhexyl)thio)‐4′‐fluorothiophen‐2′‐yl)benzo[1,2‐b:4,5‐b′]dithiophene‐2,6‐diyl)‐alt‐1,3‐bis(thiophen‐2‐yl)‐5,7‐bis(2‐ethylhexyl)benzo[1,2‐c:4,5‐c′]dithiophene‐4,8‐dione (PBDTT‐SF‐BDD), namely, via an advantageous and synthetically economic route for the key monomer are reported herein. Synergistic effects of fluorination and alkylthiolation on BDT moieties are discussed in detail, which is based on the superior balance between high V_oc and large J_sc when PBDTT‐SF‐TT/PC₇₁BM and PBDTT‐SF‐BDD/PC₇₁BM solar cells present their high V_oc as 1.00 and 0.97 V (associated with their deep highest occupied molecular orbital level of ?5.54 and ?5.61 eV), a moderately high J_sc of 14.79 and 14.70 mA cm^?2, and thus result a high power conversion efficiency of 9.07% and 9.72%, respectively. Meanwhile, for PBDTT‐SF‐TT, a very low energy loss of 0.59 eV is pronounced, leading to the promisingly high voltage, and furthermore performance study and morphological results declare an additive‐free PSC from PBDTT‐SF‐TT, which is beneficial to practical applications.     

Comparative demography of three coexisting Acer species in gaps and under closed canopy

Questions: 1. Is there a trade‐off between gap dependency and shade tolerance in each of the life‐history stages of three closely related, coexisting species, Acer amoenum (Aa), A. mono (Am) and A. rufinerve (Ar)? 2. If not, what differences in life‐history traits contribute to the coexistence of these non‐pioneer species? Location: Ogawa Forest Reserve, a remnant (98 ha), species‐rich, temperate deciduous forest in central Japan (36°56’ N, 140°35’ E, 600 ‐ 660 m a.s.l.). Methods: We estimated the demographic parameters (survival, growth rate and fecundity) by stage of each species growing in gaps and under closed canopy through observations of a 6‐ha permanent plot over 12 years. Population dynamics were analysed with stage‐based matrix models including gap dynamics. Results : All of the species showed high seedling and sapling survival rates under closed canopies. However, demographic parameters for each growth stage in gaps and under closed canopies revealed inter‐specific differences and ontogenetic shifts. The trade‐off between survival in the shade and growth in gaps was detected only at the small sapling stage (height < 30 cm), and Ar had the highest growth rate both in the shade and in the gaps at most life stages. Conclusions: Inter‐specific differences and ontogenetic shifts in light requirements with life‐form differences may contribute to the coexistence of the Acer species in old‐growth forests, with Aa considered a long‐lived sub‐canopy tree, Am a long‐lived canopy tree, and Ar a short‐lived,‘gap‐phase’ sub‐canopy tree.     

Vector competence of stable flies, Stomoxys calcitrans L. (Diptera: Muscidae), for Enterobacter sakazakii.

Enterobacter sakazakii is an opportunistic food-borne pathogen causing meningitis, enterocolitis, and sepsis, primarily in immunocompromised infants. It has been suggested that stable flies, Stomoxys calcitrans L., are a vector/reservoir of this pathogen. In this study, we assessed a) vector competence of adult stable flies (SF) for E. sakazakii, b) effect of E. sakazakii on SF development, and c) survival of E. sakazakii during SF development and colonization of the digestive tract of newly-emerged flies. Our data show that in the colony, adult SF can maintain E. sakazakii for at least 20 days regardless of the food source (blood or sugar) and contaminate the food source. The concentration of the pathogen per individual SF ranged from 1.8 x 10(5) to 6.4 x 10(6) CFU. E. sakazakii supported development of immature SF in sterilized cattle manure and sterilized artificial medium (78.3% and 76.7% SF survival to adult stage, respectively). In addition, E. sakazakii survived during SF development and colonized the gut of emerging adult SF but only when SF larvae were maintained on sterilized cattle manure inoculated with E. sakazakii (12.8% prevalence in adult SF) and on the sterile artificial medium with E. sakazakii (21.7% prevalence in adult SF). E. sakazakii was not recovered from flies or the substrate when larvae were reared on cattle manure with a non-sterilized complex microbial community plus the E. sakazakii inoculum. This study shows that SF adults have a potential to carry E. sakazakii for an extended period of time. E. sakazakii supports SF development and can survive during SF pupation and then colonize the gut of newly-emerged flies.     

Changes in Health State Utilities With Changes in Body Mass in the Diabetes Prevention Program

Health utilities are measures of health‐related quality of life (HRQL) used in cost‐effectiveness research. We evaluated whether changes in body weight were associated with changes in health utilities in the Diabetes Prevention Program (DPP) and whether associations differed by treatment assignment (lifestyle intervention, metformin, placebo) or baseline obesity severity. We constructed physical (PCS‐36) and mental component summary (MCS‐36) subscales and short‐form‐6D (SF‐6D) health utility index for all DPP participants completing a baseline 36‐item short form (SF‐36) HRQL assessment (N = 3,064). We used linear regression to test associations between changes in body weight and changes in HRQL indicators, while adjusting for other demographic and behavioral variables. Overall differences in HRQL between treatment groups were highly statistically significant but clinically small after 1 year. In multivariable models, weight change was independently associated with change in SF‐6D score (increase of 0.007 for every 5 kg weight loss; P < 0.001), but treatment effects independent of weight loss were not. We found no significant interaction between baseline obesity severity and changes in SF‐6D with changes in body weight. However, increases in physical function (PCS‐36) with weight loss were greater in persons with higher baseline obesity severity. In summary, improvements in HRQL are associated with weight loss but not with other effects of obesity treatments that are unrelated to weight loss. Although improvements in the SF‐6D did not exceed commonly reported thresholds for a minimally important difference (0.04), these changes, if causal, could still have a significant impact on clinical cost‐effectiveness estimates if sustained over multiple years.     

Betaine attenuates Alzheimer‐like pathological changes and memory deficits induced by homocysteine

Hyperhomocysteinemia (Hhcy) may induce memory deficits with β‐amyloid (Aβ) accumulation and tau hyperphosphorylation. Simultaneous supplement of folate and vitamin B12 partially restored the plasma homocysteine level and attenuated tau hyperphosphorylation, Aβ accumulation and memory impairments induced by Hhcy. However, folate and vitamin B12 treatment have no effects on Hhcy which has the methylenetetrahydrofolate reductase genotype mutation. In this study, we investigated the effects of simultaneous supplement of betaine on Alzheimer‐like pathological changes and memory deficits in hyperhomocysteinemic rats after a 2‐week induction by vena caudalis injection of homocysteine (Hcy). We found that supplementation of betaine could ameliorate the Hcy‐induced memory deficits, enhance long‐term potentiation (LTP) and increase dendritic branches numbers and the density of the dendritic spines, with up‐regulation of NR1, NR2A, synaptotagmin, synaptophysin, and phosphorylated synapsin I protein levels. Supplementation of betaine also attenuated the Hcy‐induced tau hyperphosphorylation at multiple AD‐related sites through activation protein phosphatase‐2A (PP2A) with decreased inhibitory demethylated PP2A_C at Leu309 and phosphorylated PP2A_C at Tyr307. In addition, supplementation of betaine also decreased Aβ production with decreased presenilin‐1 protein levels. Our data suggest that betaine could be a promising candidate for arresting Hcy‐induced AD‐like pathological changes and memory deficits.     

Comparison of different protein precipitation and solid‐phase extraction protocols for the study of the catabolism of peptide drugs by LC‐HRMS

LC‐HRMS‐based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent‐related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid‐phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP‐2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (H_R) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed‐mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP‐2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the H_R and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.     

CXCR4‐SF1 bifunctional adipose‐derived stem cells benefit for the treatment of Leydig cell dysfunction‐related diseases

Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29⁺ CD44⁺ CD34^? CD45^? ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.     

A new method for the production of sterile colonies of Lucilia sericata

Maggot debridement therapy (MDT) refers to the use of blowfly larvae to clean or debride an infected wound. Most commonly, larvae of Lucilia sericata (Meigen) (Diptera: Calliphoridae) are used, and are sterilized prior to use to ensure no further bacterial infections are introduced during treatment. Current methods sterilize eggs from laboratory‐reared blowfly colonies, after which sterile early second instar maggots can be provided to hospitals for use in treatment. Maggots not required for treatment are used for colony regeneration, in which sterility is not maintained. The ability to maintain sterility beyond this would allow further research into fly–bacteria interactions and the effects of different bacteria on the blowfly lifecycle. This study aimed to produce a colony of sterile adults, using current egg sterilization practice, but maintaining sterility through to pupation and emergence. The production of a sterile colony allows further research into the impact of bacteria on fly development and survival. Eggs were placed on a sterile food source within autoclaved plant tissue culture containers to allow growth under sterile conditions. Nutrient agar plating of sterilized and non‐sterilized eggs, larvae and adults (post‐emergence), as well as the pupation medium and feed source in nutrient broth confirmed the aerobic sterility of all samples involved. The lifecycle of L. sericata was successfully completed through pupation to emergence with no effects on lifespan or oviposition by the newly emerged, sterile adult colony.     

Accumulation of heptaprenyl diphosphate sensitizes Bacillus subtilis to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter

Heptaprenyl diphosphate (C₃₅‐PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C₃₅‐PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C₅₅‐PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C₃₅‐PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C₃₅‐PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C₃₅‐PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepT or by supplementation with 1,4‐dihydroxy‐2‐naphthoate, a co‐substrate with C₃₅‐PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C₃₅‐PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C₃₅‐PP inhibits both BcrC (a C₅₅‐PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C₅₅‐PP, rather than the antibiotic across the membrane.