Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars

Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.     

Grooming Circadian Rhythmicity,Progesterone Levels and Partner Preference of the Reproductive Pair of a Captive Common Marmoset (Callithrix jacchus) Family Group during Pregnancy and after Parturition

One family group of common marmosets (Callithrix jacchus) maintained in captivity under natural environmental conditions (Natal, Brazil, 05°46'S, 35°12'W) was observed by focal sampling at two non-consecutive days per week during two non-consecutive pregnancies (pregnancy 1: the last 8 fortnights before parturition; pregnancy 2: the last 5 ones) and after parturition (4 fortnights for both). The hourly percentiles of the duration of self-grooming and allogrooming interactions of the reproductive pair were submitted to Cosinor analysis for the 24-h period (p ≤ 0.05). The reproductive female's progesterone was monitored by fecal collection twice a week between 7:00 h to 9:00 h. Levels of progesterone were determined by ELISA method. During pregnancy 1, all interactions of the reproductive pair showed a 24-h statistically significant period. However, after parturition this periodicity is not present in some behaviors of the reproductive male. This animal presents the 24-h periodicity only toward to the reproductive female, the subadult son and to the newborns. Hormonal levels of the reproductive female showed isolated increases above 100 ng/g at the second and third fortnight after parturition, but did not characterize ovulation. Between pregnancy 1 and 2, the family was increased by the birth of four animals. During pregnancy 2, a statistically significant 24-h period was find only in self-grooming of the reproductive male and female, and in the interactions between the reproductive pair. Also, in the interactions of the reproductive male with only one of the juvenile son. After parturition the reproductive female ovulated at the second fortnight and became pregnant again. At this time, those interactions still have a 24-h period, except in grooming directed to the juvenile son by the reproductive male. As in the first post-partum, grooming directed to the newborns showed statistically significant 24-h period. These results suggest that the reproductive pair performs 24-h rhythmic grooming interactions only with some animals, supposed to be the preferential grooming partners. Furthermore, the temporal grooming expression in common marmosets may be affected by the reproductive condition (progesterone levels) of the reproductive female, by the care of newborn after parturition and the group size.     

Integrated diversity analysis of the microbial community in a reverse osmosis system from a Brazilian oil refinery

Oil refineries are known for the large volume of water used in their processes, as well as the amount of wastewater generated at the end of the production chain. Due to strict environmental regulations, the recycling of water has now become a viable alternative for refineries. Among the many methods available to treat wastewater for reuse, the use of membranes in reverse osmosis systems stands out due to several economic and environmental benefits. However, these systems are vulnerable to contamination and deposition of microorganisms, mainly because of the feedwater quality. In this study, the microbial diversity of feedwater and reverse osmosis membranes was investigated using a combination of culture-dependent and culture-independent methods in order to characterize the microorganisms colonizing and deteriorating the membranes. In total, 37 bacterial isolates, 17 filamentous fungi and approximately 400 clones were obtained and analyzed. Among the bacterial genera identified, the most represented were Sphingobium, Acidovorax, Microbacterium, Rhizobium and Shinella. The results revealed genera that acted as candidate key players in initial biofilm formation in membrane systems, and provided important information concerning the microbial ecology of oligotrophic aquatic systems.     

Crystal structure of the lectin of Camptosema pedicellatum: implications of a conservative substitution at the hydrophobic subsite

Lectins have been used as models for studies of the molecular basis of protein-carbohydrate interaction and specificity by deciphering codes present in the glycan structures. The purpose of the present study was to purify and solve the complete primary and crystal structure of the lectin of Camptosema pedicellatum (CPL) complexed with 5-bromo-4-chloro-3-indolyl-α-d-mannose (X-Man) using tandem mass spectrometry. CPL was purified by single-step affinity chromatography. Mass spectrometry findings revealed that purified CPL features a combination of chains weighing 25,298 ± 2 (α-chain), 12,835 ± 2 (β-chain) and 12,481 ± 2 Da (γ-chain). The solved crystal structure of CPL features a conservative mutation in the hydrophobic subsite, a constituent of the carbohydrate recognition domain (CRD), indicating the relevance of hydrophobic interactions in the establishment of interactions with carbohydrates. The substitution and the analysis of the interactions with X-Man also revealed that the hydrophobic effect caused by a minor change in the hydrophobic subsite interferes in the formation of H-bonds due to the reorientation of the indolyl group in the CRD.     

叶绿素结合蛋白CP24介导光照响应基因StRSM 1调控叶绿素积累

单MYB转录因子成员RSM(RADIALIS-like SANT/MYB)突变,倒置黑暗诱导的叶绿素减少,原因有待确定;为了揭示RSM如何调控叶绿素积累,本研究运用基因工程途径,获得了普通烟草和马铃薯体内抑制和过量表达StRSM 1阳性转化株系,测定了阳性转化株系的叶绿素积累等生理表型;结果显示,普通烟草和马铃薯体内抑制RSM 1表达,显著增加了叶绿素积累,叶色随之加深;RSM 1过量表达,显著减少叶绿素积累,叶色变浅。叶绿素代谢相关基因表达测定结果显示,StRSM 1过量表达增加了黑暗下叶绿素结合蛋白CP24基因的表达,改变了其表达模式。以上结果表明,转录因子StRSM 1响应光照反向调控叶绿素积累,叶绿素结合蛋白CP24参与了StRSM 1对叶绿素积累的调控。结果有助于进一步明确RSM 1如何响应光照和深刻理解RSM 1参与的光照响应。     

Rab35 and glucocorticoids regulate APP and BACE1 trafficking to modulate Aβ production

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms, including overproduction of toxic amyloid-β (Aβ) peptides and intraneuronal accumulation of hyperphosphorylated Tau protein. The latter is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in Aβ overproduction remains an open question. Here, we find that hippocampal Rab35 levels are decreased not only by stress/GC but also by aging, another AD risk factor. Moreover, we show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking steps for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Finally, we demonstrate that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by high GC levels. These studies identify Rab35 as a key regulator of APP processing and suggest that its downregulation may contribute to stress-related and AD-related amyloidogenesis.Subject terms: Cellular neuroscience, Alzheimer''s disease     

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K _b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K _b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.     

A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field‐collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real‐time polymerase chain reaction (RT‐PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme‐linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP‐ELISA). However, because of the relatively high costs associated with equipment and reagents, RT‐PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA‐based nested PCR protocol, modified from a loop‐mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP‐ELISAs in field‐collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4‐fold higher sensitivity than the CSP‐ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium‐positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.     

Proteome analysis of Nelore bull (Bos taurus indicus) seminal plasma

The Nelore bull (Bos taurus indicus) seminal plasma proteome was analyzed by MALDI-TOF MS and two-dimensional gel electrophoresis. A total of 260 spots were visualized in the 2-DE gel (pI range 3-10) and 13 spots could be identified by peptide mass fingerprinting corresponding to 11 different polypeptides. The results allowed the creation of the first proteomic map of Bos taurus indicus seminal plasma. The roles of the identified proteins in the bull seminal plasma are discussed.     

Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing‐fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.