Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Role of 𝛃 1-4 linked polymers in the biofilm structure of marine Pseudomonas sp. CE-2 on 304 stainless steel coupons

Pseudomonas sp CE-2 cells attach and form biofilms on 304-stainless steel (SS) coupons. A series of experiments were carried out in order to understand the role of exopolysaccharides (EPS) in the formation and maintenance of CE-2 biofilms on SS coupons. The biofilm density and EPS concentration increased over the period of incubation and the highest values for both were recorded after 72 h. Calcofluor and the lectin concanavalin A (Con A) showed a positive interaction with 72-h old biofilms, indicating the presence of β 1-4 linked polymers, and α-d-glucose and α-d-mannose in the biofilm matrix of CE-2. When the CE-2 cells were grown in the presence of calcofluor (200 μg ml^?1), biofilm formation was significantly reduced (～85%). Conversely, the lectins Con A or WGA did not influence the CE-2 biofilms on the SS coupons. Furthermore, treatment with cellulase, an enzyme specific for the degradation of β 1-4 linked polymers, removed substantial amounts of CE-2 biofilm from SS coupons. These results strongly suggest the involvement of β 1-4 linked polymers in the formation and maintenance of Pseudomonas sp. CE-2 biofilms on SS coupons.     

A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate

Phytase (EC 3.1.3.–) hydrolyzes phytate (IP₆) present in cereals and grains to release inorganic phosphate (P_i), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P_i liberated from IP₆. This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP₆–lysozyme as a substrate. The assay is based on the principle that IP₆ forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP₆ in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P_i from IP₆. This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl₂, and phosphate, showed a concentration-dependent interference.     

PIWI-interacting RNA 39980 promotes tumor progression and reduces drug sensitivity in neuroblastoma cells

Neuroblastoma (NB) is the leading pediatric cancer known for its heterogeneity and clinical aggressiveness leading to chemoresistance. Recent evidence in small RNA research has led to the discovery of PIWI-interacting RNAs (piRNAs) which work in an orchestrated fashion to modulate gene expression both in homeostatic conditions and abnormalities like cancer including NB. This study aims to decipher the possible role of a repeat-derived piRNA, piR-39980 (identified from our previous piRNA profiling study in human NB cell lines) in tumorigenesis of NB cells. piR-39980, overexpressed in NB cells act as an oncopiR and promotes tumor progression, while its inhibition resulted in reduced viability, invasion as well as the migration of IMR-32 cells. Interestingly, we observed that inhibition of piRNA induces senescence of NB cells without affecting the classical apoptosis pathway by modulating the expression of JAK3 through target binding. In addition, piR-39980 was found to desensitize the effect of doxorubicin and inhibit drug-induced apoptosis. Overall, we report piR-39980, as the first oncopiR which might serve as a novel therapeutic target for this malignancy.     

Subcellular dynamics of variants of SG2NA in NIH3T3 fibroblasts

SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)‐tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3β‐ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP‐tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP‐tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation‐dephosphorylation. The inhibition of GSK3β by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP‐tagged 35, 52, and 87 kDa, inhibition of GSK3β caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3β by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular‐signal‐regulated‐kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3β, and ERK. Taken together, the variants of SG2NA are modulated by the kinase‐phosphatase network in a similar but characteristic manner.     

Neuroprotective effects of mitochondria‐targeted curcumin against rotenone‐induced oxidative damage in cerebellum of mice

The present study investigated the protective effect of curcumin and mitochondrial‐targeted curcumin (MTC) in rotenone‐induced cerebellar toxicity in mice. Treatment of rotenone in mice significantly shortened the stride length for both forelimb and hind‐limb and increased fore‐paws and hind‐limb base width. Co‐treatment of curcumin and MTC with rotenone improved the walking pattern. A significant increase in lipid peroxidation, nitric oxide and decreased activity of AChE, reduced glutathione, superoxide dismutase and catalase were observed in rotenone‐treated mice while co‐treatment of curcumin and MTC with rotenone significantly increased AChE activity and protected against rotenone‐induced oxidative damage. Rotenone exposed mice showed irregular, damaged Purkinje cells and perineuronal vacuolation while co‐treatment of curcumin and MTC with rotenone protected against rotenone‐induced cellular damage in these cells. The result exhibits that both curcumin and MTC showed protective effects against rotenone‐induced cerebellar toxicity in mice and MTC is more effective than curcumin.     

Causes of slowing‐down seasonal CO2 amplitude at Mauna Loa

Changing amplitude of the seasonal cycle of atmospheric CO₂ (SCA) in the northern hemisphere is an emerging carbon cycle property. Mauna Loa (MLO) station (20°N, 156°W), which has the longest continuous northern hemisphere CO₂ record, shows an increasing SCA before the 1980s (p < .01), followed by no significant change thereafter. We analyzed the potential driving factors of SCA slowing‐down, with an ensemble of dynamic global vegetation models (DGVMs) coupled with an atmospheric transport model. We found that slowing‐down of SCA at MLO is primarily explained by response of net biome productivity (NBP) to climate change, and by changes in atmospheric circulations. Through NBP, climate change increases SCA at MLO before the 1980s and decreases it afterwards. The effect of climate change on the slowing‐down of SCA at MLO is mainly exerted by intensified drought stress acting to offset the acceleration driven by CO₂ fertilization. This challenges the view that CO₂ fertilization is the dominant cause of emergent SCA trends at northern sites south of 40°N. The contribution of agricultural intensification on the deceleration of SCA at MLO was elusive according to land–atmosphere CO₂ flux estimated by DGVMs and atmospheric inversions. Our results also show the necessity to adequately account for changing circulation patterns in understanding carbon cycle dynamics observed from atmospheric observations and in using these observations to benchmark DGVMs.