Crude Preparations Research Articles

Analysis of chemical properties for biodiesel derived from crude palm oil

AbstractHigh biodiesel purity (98.86% to 99.54%) has been achieved using cooking oil, which undergoes various purification stages before being sold. This inherent purity contributes to the high biodiesel purity produced during solvent‐aided crystallization (SAC). This study aims to investigate the performance of SAC using unpurified oil as the feedstock for crude biodiesel preparation. Crude palm oil (CPO) was used as the feedstock, and the effects of crystallization temperature (6, 8, 10, 12, and 14°C), crystallization time (20, 25, 30, 35, and 40 min), and shaking speed (32, 43, 61, 71, and 84 rpm) were measured. The highest biodiesel purity achieved was 99.05% at 6°C crystallization temperature, 30 min crystallization time, and medium shaking speed. Additionally, all samples met international standards EN 14214 and ASTM D6751 for chemical properties, including iodine and acid value analysis. These results suggest that SAC is an effective method for producing high‐quality biodiesel from less refined feedstock, potentially lowering production costs and expanding the range of viable raw materials for biodiesel production.

Use of thermophilic bacteria to utilize sugarcane bagasse: Efficient cellulase production, saccharification, and hydrolysate applications

Sugarcane bagasse (SB) a renewable and abundant source of rich fermentable sugars has been reported extensively to produce microbial enzymes and other valuable products. However, most of the SB-based processes require its pretreatment which is an additional step incurring time and cost. In this study, fermentation of SB by bacterial strains without any pretreatment was carried out to obtain crude cellulase preparation. Enzyme mediated saccharification of SB was carried out and the hydrolysate was analyzed for the presence of reducing sugars and free radical scavenging activity. It was found that 65.86, 42.03 and 88.8 IU mL−1 endoglucanase was produced by Brevibacillus borstelensis UE10, Neobacillus sedimentimangrovi UE25 and B. borstelensis UE27, respectively by fermenting SB. Crude cellulase preparation of UE10, UE25, and UE27 was used to saccharify SB which resulted in 1.14, 1.47, and 1.58 mg mL−1 reducing sugars and 1.08, 0.57, and 1.5 mg mL−1 glucose, respectively. Moreover, the hydrolysate also had radical scavenging activity indicating about its possible application in animal feed. The maximum (35.71 %) amount of bioethanol was produced when UE25 fermented the hydrolysate of SB. The strains UE10 and UE27 produced 31.16 and 27.77 % bioethanol, respectively. Furthermore, Fourier transform infrared spectroscopy, scanning electron microscopy and gravimetric analysis showed the impact of cellulase on structure of SB. Hence, this work expands the understanding of the potential of thermophilic bacterial strains to utilize agricultural waste for the synthesis of valuable products.

Iron content of commercial mucin contributes to compositional stability of a cystic fibrosis airway synthetic microbiota community.

In vitro culture models of mucosal environments are used to elucidate the mechanistic roles of the microbiota in human health. These models often include commercial mucins to reflect the in-situ role of mucins as an attachment site and nutrient source for the microbiota. Two types of mucins are commercially available: porcine gastric mucin (PGM) and bovine submaxillary mucin (BSM). These commercial mucins have been shown to contain iron, an essential element required by the microbiota as a co-factor for a variety of metabolic functions. In these mucin preparations, the concentration of available iron can exceed physiological concentrations present in the native environment. This unexpected source of iron influences experimental outcomes, including shaping the interactions between co-existing microbes in synthetic microbial communities used to elucidate the multispecies interactions within native microbiota. In this work, we leveraged the well-characterized iron-dependent production of secondary metabolites by the opportunistic pathogen Pseudomonas aeruginosa to aid in the development of a simple, low-cost, reproducible workflow to remove iron from commercial mucins. Using the mucosal environment of the cystic fibrosis (CF) airway as a model system, we show that P. aeruginosa is canonically responsive to iron concentration in the chemically defined synthetic CF medium complemented with semi-purified PGM, and community composition of a clinically relevant, synthetic CF airway microbial community is modulated, in part, by iron concentration in PGM.

Comparative analysis of microbial diversity and functional characteristics of low-temperature Daqu from different regions in China

Baijiu is a unique alcoholic beverage in China, prepared through a traditional solid-state fermentation process. Daqu, recognized as the earliest form of crude enzyme preparation, plays a crucial role in determining the quality of Baijiu. Due to the enzymatic process derived from natural fermentation, the differences between regions were expected to occur. To compare regional-difference in microbial diversity and the functional profile of Daqu, metagenomic sequencing, physicochemical analysis, and electronic sensory evaluation were utilized. The results showed that bacteria and fungi were the main microbes in low-temperature Daqu (LTD), and their ratio of abundance was approximately 9:1. Streptomyces (20.27 %) and Bacillus licheniformis (15.28 %) emerged as the most dominant microbes in LTD at the genus and species levels, respectively. The overall microbial communities and functional profiles of Daqu between the two regions exhibited significant differences (P < 0.05). Bacillus licheniformis was significantly enriched in LTD from Taiyuan (P < 0.05) and mainly contributed to the saccharifying power, fermenting power, esterifying power, and liquefying power of LTD, demonstrating its important role in maintaining LTD quality. LTD from Taiyuan had a significantly higher fermenting power, esterifying power, and liquefying power than LTD from Suizhou (P < 0.05). In addition, a higher abundance of genes responsible for lactate production was detected in LTD from Suizhou, whereas genes associated with acetate production were enriched in LTD from Taiyuan. Collectively, these findings indicated that lactic acid bacteria may play a more important role in LTD from Suizhou, whereas acetate-producing bacteria may have a greater contribution to LTD from Taiyuan. This study provides a reference for microbial regulation during the production process of Daqu, and control of quality and traceability of the origin of Daqu.

Enhanced decision technique for optimized crude oil pretreatment under disc spherical fuzzy Aczel Alsina aggregation information

Crude oil, the backbone of modern industry, holds unparalleled significance as a global energy cornerstone. Unlocking its potential hinges on effective pretreatment techniques, ensuring purity, and maximizing efficiency. This study extends the established Spherical Fuzzy Set paradigm to explore the domain of Disc Spherical Fuzzy Sets (D-SFSs) in critical decision-making for crude oil preparation. Investigating D-SFSs within the Aczel Alsina norm, the research employs comparison rules, conversion rules, and distance metrics. Primary operations of the Aczel Alsina norm in D-SFSs are examined, laying the groundwork for introducing unique aggregation operations within this framework. The paper’s primary aim is to propose a hybrid method, termed MEREC-SWARA-MARCOS-D-SFSs Multiple Attribute Group Decision Making, which integrates the aforementioned aggregation procedures. A case study on crude oil pretreatment validates the effectiveness of the proposed method. Furthermore, a comprehensive comparison with CoCoSo underscores the reliability of the method. This study represents a significant stride in enhancing decision-making by providing a robust framework to tackle complex situations, particularly in the critical domain of crude oil pretreatment.

Enzyme purification and sustained enzyme activity for pharmaceutical biocatalysis by fusion with phase-separating intrinsically disordered protein.

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.

Combined use of versatile peroxidase and aryl alcohol oxidase of Pleurotus eryngii to decolorize melanin on the skin

Melanin plays an important role in protecting skin cells from harmful UV radiation, but its uneven pigmentation in the skin sometimes demands cosmetic resolution. As a safe and effective way of evening the skin tone, enzymatic decolorization of melanin at the stratum corneum has been proposed. In this regard, the use of lignin peroxidase, in the presence of hydrogen peroxide and veratryl alcohol, an electron mediator, has been explored. Here, we first show that versatile peroxidase (VP) and aryl alcohol oxidase (AAO) purified from the Pleurotus eryngii liquid culture can be used to decolorize melanin without exogenous hydrogen peroxide. This resembles the oxidative degradation pathway of lignin in nature; AAO generates hydrogen peroxide from veratryl alcohol, and VP utilizes generated hydrogen peroxide while using veratryl alcohol as a mediator. We further explored the use of POX_Pe, the crude peroxidase preparation of the Pleurotus eryngii liquid culture, for melanin decolorization because of its better stability. Using POX_Pe and veratryl alcohol, over 60 % of melanin decolorization was obtained in 1 h in the absence of exogenous hydrogen peroxide addition. Furthermore, POX_Pe could decolorize melanin in a 3D human pigmented epidermis model, demonstrating its possible applications in cosmetics.

Valorization of rice straw and vascular aquatic weeds for sustainable prebiotic hemicellulosic autohydrolysate production: Extraction, characterization and fermentability.

This study describes the extraction and characterization of the hemicellulosic autohydrolysates (HAHs) derived from rice straw (RS) and vascular aquatic weeds like Typha angustifolia (TA) and Ceretophyllum demersum (CD). It further explores their capacity to sustain the proliferation of selected lactic acid bacteria (i.e., prebiotic activity) isolated from milk samples. To fractionate HAH from RS, TA and CD hot water extraction (HWE) method was used and RS, TA, and CD biomasses yielded 6.8, 4.99 and 2.98% of HAH corresponding to the hemicellulose extraction efficiencies of 26.15 ± 0.8%, 23.76 ± 0.6%, and 18.62 ± 0.4% respectively. The chemical characterization of HAH concentrates through HPLC showed that they comprised galactose, arabinose, xylose and glucose. The total phenol content of the RS, TA and CD-derived HAH concentrates were 37.53, 56.78 and 48.08mg GAE/g. The five lactic acid bacteria (LAB) isolates Q1B, Q2A, Q3B, G1C and G2B selected for prebiotic activity assays generated mixed responses with the highest growth in RS-HAH for Q2A and the least in TA-HAH for Q3B. Further, the isolates Q2A, Q3B, G1C, and G2B, which showed the highest growth performance, were identified through MALDI-TOF and 16S rRNA sequencing as Lactobacillus brevis. All the tested LAB isolates showed diauxic growth in crude HAH preparations to maximize the utilization of carbon resources for their proliferation. This suggests that the selected LAB isolates are efficient degraders of hemicellulosic sugars. This paves the way for the valorization of lignocellulosic biomass to produce prebiotic hemicellulosic autohydrolysate and consequently enhances environmental sustainability by improving resource efficiency.

Neutralization of ricin toxin on building interior surfaces using liquid decontaminants.

Ricin is a highly toxic protein, capable of inhibiting protein synthesis within cells, and is produced from the beans of the Ricinus communis (castor bean) plant. Numerous recent incidents involving ricin have occurred, many in the form of mailed letters resulting in both building and mail sorting facility contamination. The goal of this study was to assess the decontamination efficacy of several commercial off-the-shelf (COTS) cleaners and decontaminants (solutions of sodium hypochlorite [bleach], quaternary ammonium, sodium percarbonate, peracetic acid, and hydrogen peroxide) against a crude preparation of ricin toxin. The ricin was inoculated onto four common building materials (pine wood, drywall joint tape, countertop laminate, and industrial carpet), and the decontaminants were applied to the test coupons using a handheld sprayer. Decontamination efficacy was quantified using an in-vitro cytotoxicity assay to measure the quantity of bioactive ricin toxin extracted from test coupons as compared to the corresponding positive controls (not sprayed with decontaminant). Results showed that decontamination efficacy varied by decontaminant and substrate material, and that efficacy generally improved as the number of spray applications or contact time increased. The solutions of 0.45% peracetic acid and the 20,000-parts per million (ppm) sodium hypochlorite provided the overall best decontamination efficacy. The 0.45% peracetic acid solution achieved 97.8 to 99.8% reduction with a 30-min contact time.

A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography.

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 μg or less of purified products, this method can purify ∼100-1000 μg of DNA nanostructures in less than 30 min, with the overall collection yield of 50-70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions.

Valorization of Chicken Viscera as Natural Raw Material Source: Application to Hydrolysis of Fatty Esters for Sewage Treatment.

"Chicken viscera" constitutes very abundant domestic wastes interestingly investigated in the present paper. The efficiency of this crude slaughter co-product of high protein component, as biocatalyst, for the hydrolysis of fatty acid esters was reported and that, without any pre-treatment. The crude chicken intestine powder (CIP) has shown a high reactivity for the hydrolysis of fatty esters. Two biocatalyst preparations were independently explored for the bioresolution of sec-phenyl alkyl carbinol esters: the CIP preparation and the crude chicken intestine acetone powder CIAP preparation. The last one has shown good catalytic activity during the bio-hydrolysis in biphasic medium. Furthermore, the direct hydrolysis of milk fat using CIAP (500mg) reveals the elimination of fats present in 50ml of treated milk. These results open up very interesting prospects for the use of this biowaste for the treatment of milk fat.

Application of a crude digestive proteases preparation to improve the ripening of marinated fillets from low-technological value Baltic herring (Clupea harengus membras L.).

As a result of climate change (reduced the oxygen content and food available in the waters) and overfishing, ever larger batches of the herring catch are classified as low-value fish and used for feedstuff or canned food production. Fast and complete ripening of marinated fillets, especially from low-value Baltic herring, poses a problem because of the low muscle protease activity and changes in muscle tissue proteins. For the first time, a crude digestive proteases preparation (CDPP) was obtained from herring viscera using a two-stage method consisting of ethanol extraction and then salt precipitation. CDPP had a reduced hemoglobin content, with optimum activity at pH 7.5-8.8 or 60-120 g kg-1 NaCl. At pH 4-5, it still exhibited 24-68% of proteolytic activity. CDPP was used for 4-24 h of brining of fresh and frozen-thawed fillets or injection of fresh fillets before marinating. CDPP-brining increased especially cathepsin D and carboxypeptidase A activities, whereas it decreased cathepsin B and L activities in the marinades. CDPP-brining mitigated the negative effect of freezing-thawing on mass-yield, protease activity, protein hydrolysis, texture profile, colour and sensory quality of the marinated fillets. CDPP-injection was found to be the best method because it increased mass-yield and ripeness of the marinated fillets to a greater extent than CDPP-brining did. The marinades from the CDPP-treated fillets had no bitter taste as a result of the presence of hemoglobin or chymotrypsin, and there were no results indicating lipid oxidation. The application of CDPP in marinating technology is a viable approach to enable the use of low-value herring in food production, shorten the marinating time, and improve the ripeness and sensory quality of meat. © 2024 Society of Chemical Industry.

Effect of Natural IAA Produced by Streptomyces Strain ACM37 on Seed Germination and Seedling Growth

&#x0D; &#x0D; &#x0D; Biofertilizers play an important role in sustainable agriculture by improving soil fertility, crop tolerance and crop productivity. In this context, exploitation of plant growth promoting rhizobacteria with various beneficial properties is challenging. This study aimed to investigate the plant growth promoting activity of indole-3-acetic acid (IAA)-producing actinomycetes strain, Streptomyces ACM37 on the seed germination and seedling growth at early stage. The ACM37 was grown in starch-casein broth for 7 days at 28° C, 80 rpm and supplemented with 50 mg/l L- tryptophan to induce IAA production. After 7 days, IAA concentration in cell-free culture broth was quantified by following the Salkowski's method and it was 50 mg/l. Seed germination and seedling growth of cowpea (Vigna unguiculata) and rice (Oryza sativa L.) at different concentrations of IAA were estimated as % seed germination, number of roots, primary root length and length of hypocotyl. Initially, seeds were surface sterilized with 10% (v/v) NaOCl and soaked in 10, 20, 30, 40 and 50 mg/l crude IAA preparations for 1 h at 30° C. As controls, seeds were soaked in sterilized distilled water (SDW) and 50 mg/l synthetic IAA. Ten seeds per each treatment with three replicates were placed on 0.8% (w/v) water-agar in Petri plates and incubated in dark at 30° C. The % seed germination of ACM37-IAA-treated cowpea and rice seeds was enhanced at all concentrations compared to SDW and synthetic IAA treatments after 3 days. The highest 100% germination of cowpea was given at 10 mg/l IAA while it was 96.67±3.33% in&gt;30 mg/l IAA in rice. Similarly, there was a significant (P&lt;0.05) increase in primary root length and number of lateral roots in cowpea at 10 mg/l of ACM37-IAA. Overall, ACM37 showed significant plant growth promotion in cowpea at low concentration (10 mg/l) of IAA whereas there was a significant (P&lt;0.05) reduction at high concentrations (&gt;10 mg/l). On the other hand, there was no apparent increase in all growth parameters except seed germination in rice at all concentrations. It appears that high concentrations of ACM37-IAA may be required to promote plant growth in rice. Overall results highlight that plant growth promotion activity of ACM37 varies at different IAA concentrations and different plant species, probably between monocot and dicot species. Therefore, ACM37 has potential application as a plant growth promoting agent in biofertilizers and plant growth inhibiting agent in bioherbicides at certain IAA concentrations.&#x0D; Keywords: Actinomycetes, Biofertilizer, Bioherbicide, IAA&#x0D; &#x0D; &#x0D;

Cepacian degrading Cytobacillus sp. strain Dbc1 with anti-biofilm activity potentiating antibiotic efficacy against Burkholderia cepacia biofilm development

Aim: Inhibition of biofilm formation in Burkholderia cepacia through enzymatic degradation of predominant exopolysaccharide (cepacian) of B. cepacia. Methodology: Cepacian was extracted from B. cepacia followed by isolation and identification of potent cepacian utilizing bacteria based on planktonic growth using cepacian as sole carbon source. Effective lyase activity (responsible for cepacian breakdown) and anti-biofilm activity of potent isolate against B. cepacia was determined. Effect of biofilm disintegration on antibiotic penetration into biofilm was detected. Lastly, inhibition of biofilm formation by crude lyase preparation of the isolate inside an external medical device was detected. Results: Cytobacillus sp. strain Dbc1 was the most potent cepacian degrading bacteria which showed significant lyase activity and reduced total biomass of both newly formed and pre-formed biofilm of B. cepacia. Cepacian degradation potentiated chloramphenicol penetration within pre-formed biofilm leading to cell mortality in B. cepacia. Cytobacillus sp. strain Dbc1significantly reduced total biomass of established biofilm inside nasal oxygen catheter. Interpretation: It can be concluded that Cytobacillus sp. strain Dbc1 significantly controlled mature B. cepacia biofilm through potent cepacian degrading and anti-biofilm activity along with better antibiotic targeting into established biofilm. Strain Dbc1 can be used to inhibit medical device- associated biofilms in vitro. Key words: Antibiotic susceptibility, Biofilm inhibition, Burkholderia cepacia, Cepacian breakdown, Multi-drug resistance

Polyphenol Oxidase Enzyme Activity of Mulberries Grown in Iğdir Ecological Conditions

The presence of polyphenol oxidase enzyme (PFO) in black and white mulberry varieties grown in Iğdır province were determined. In the study, no polyphenol oxidase enzyme was detected in the white mulberry varietie, therefore the study was conducted only with black mulberry varieties. The mulberry samples were collected from Iğdır Center, Melekli and Karakoyunlu districts during June and July, both in 2015 and 2016, when the fruits were fully ripe. Mulberries were stored at -20 °C, and the crude enzyme extract was stabilized with pH 5.0 sodium-acetate buffer containing 1% Polyethylene glycol (PEG). To weigh with a precision scale, 20 grams of frozen mulberries were taken into mortars. After the mulberries were thoroughly crushed in these mortars, 40 ml of crude enzyme extract preparation solution was added to each variety and mixed homogeneously. These mixtures were filtered through a cheesecloth and rotated at 4 °C, 10,000 rpm for 30 minutes. The obtained supernatants were used for enzyme solubilization, and the enzyme activity of the solution was determined spectrophotometrically. The PFO content depended on the genotype. There was a significant difference among the tested mulberry genotypes.

Elucidating the Structural Dynamics of Integrin α IIbβ 3 from Native Platelet Membranes By Cryo-EM Coupled with Build and Retrieve Data Processing Methodology

Background: Platelets play a critical role in physiological processes by sensing the extracellular environment through their membrane proteins. Changing the quantity or quality of these proteins can profoundly affect platelet physiology and impact health. The native membrane environment provides essential additional regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins and large macromolecules to be solved from highly purified, homogeneous samples. Our recent breakthroughs in data processing make it feasible to obtain high-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the novel “Build and Retrieve” (BaR) data processing methodology. By performing in silico purification, image sorting, and model building from large heterogenous datasets, this iterative bottom-up methodology opens new avenues for an in-depth systems biology approach with structural biology that will uncover how the native environment, including lipid and metal binding, post-translational modifications, and co-factor associations regulate platelet function at the molecular level. Method: Whole blood was collected, and the platelet membrane proteins were solubilized by 1% n-Dodecyl-β-D-Maltoside (DDM) and incorporated into lipidic nanodisc MSP1E3D1 directly from the endogenous source. After being enriched by size exclusion chromatography, the mixture of proteins at ~200 kDa was directly applied on cryo-EM grids. Data were collected on Titan Krios G3i cryogenic transmission electron microscope equipped with a BioQuantum K3 camera. All data processing was done in cryoSPARC using the BaR protocol. The near-atomic-resolution cryo-EM maps were used for protein identification using the program phenix.sequence_from_map in the PHENIX suite. The final protein models were built by Coot and refined by PHENIX. Results: We have used using cryo-EM followed by the BaR method to solve the first unmodified integrin α IIbβ 3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.76Å and 2.49Å, respectively. From these two structures, we observed the ion binding sites at the β1-domain and β-propeller domain in agreement with previous “classical” α IIbβ 3 structural studies using a purified sample. More importantly, we were able to assign endogenous N-linked glycan sites, which reflected how glycosylation naturally regulates α IIbβ 3 dynamic and function on resting human platelets. Finally, we also solved a novel third unique dimer conformation of α IIbβ 3 at 2.61Å formed by two intermediate-states of α IIbβ 3. This may indicate a previously unknown self-regulatory mechanism of α IIbβ 3 in its native environment. Conclusion: In conclusion, our data show the power of using cryo-EM with the BaR method to uncover novel structures directly from natural sources to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. A distinct advantage of the BaR method is that it is an unbiased approach for solving multiple structures from raw samples, which highlights the potential of building a protein structural atlas from platelets. These data have the potential to enrich our understanding of platelet signaling circuitry and foster the development of higher-quality diagnoses and treatments for patients with platelet disorders.

Calcium-Dependent Interaction of Nitric Oxide Synthase with Cytochrome c Oxidase: Implications for Brain Bioenergetics.

Targeted nitric oxide production is relevant for maintaining cellular energy production, protecting against oxidative stress, regulating cell death, and promoting neuroprotection. This study aimed to characterize the putative interaction of nitric-oxide synthase with mitochondrial proteins. The primary finding of this study is that cytochrome c oxidase (CCO) subunit IV (CCOIV) is associated directly with NOS in brain mitochondria when calcium ions are present. The matrix side of CCOIV binds to the N-terminus of NOS, supported by the abrogation of the binding by antibodies towards the N-terminus of NOS. Evidence supporting the interaction between CCOIV and NOS was provided by the coimmunoprecipitation of NOS from detergent-solubilized whole rat brain mitochondria with antibodies to CCOIV and the coimmunoprecipitation of CCOIV from crude brain NOS preparations using antibodies to NOS. The CCOIV domain that interacts with NOS was identified using a series of overlapping peptides derived from the primary sequence of CCOIV. As calcium ions not only activate NOS, but also facilitate the docking of NOS to CCOIV, this study points to a dynamic mechanism of controlling the bioenergetics by calcium changes, thereby adapting bioenergetics to cellular demands.

Serological analysis reveals differential antibody responses between TB patients and latently infected individuals from the TB endemic country of Mozambique.

Serological antibody profiling of tuberculosis (TB) patients and household contacts with latent TB infection (LTBI) could identify risk indicators of disease progression, and potentially also serve as an easily accessible diagnostic tool to discriminate between these two stages of Mycobacterium tuberculosis (Mtb) infection. Yet, despite significant efforts over many decades, neither application has yet fully materialised, and this is at least in part due to inconsistent and varying antibody profiles from different TB endemic regions. In this study, we conducted a retrospective exploratory analysis of serum antibodies in a cohort of active TB patients (ATB) and their interferon-gamma release assay (IGRA) positive household contacts (LTBI), as well as healthy controls (HC) from Mozambique, a country with a high TB burden from the Sub-Saharan region. Using several Mtb antigens as well as crude preparations of culture filtrate proteins (CFP) from Mtb and Bacille Calmette Guérin (BCG), we report that the most discriminatory response for TB and LTBI was observed for serum IgA antibodies to the MPT64 antigen, followed by IgG antibodies to Ag85B and CFP, with ATB patients having significantly higher levels than LTBI or BCG-vaccinated healthy controls. Conversely, sera from LTBI individuals had higher levels of IgG antibodies to the HBHA antigen than ATB. While our sample size (n = 21 for ATB, 18 for LTBI and 17 for HC) was too small to fully evaluate the diagnostic potential of these differing serological profiles, our study however preliminarily indicated high level of sensitivity (95%) and specificity (97%) of an ELISA MPT64-IgA test for discriminating TB from LTBI and healthy controls, supporting the notion that it alone, or possibly in combination with other antigens such as Ag85B or CFP could lead to development of an easily accessible diagnostic tool for TB.

Comparative analysis of nascent RNA sequencing methods and their applications in studies of cotranscriptional splicing dynamics.

High-throughput detection of nascent RNA is critical for studies of transcription and much more challenging than that of mRNA. Recently, several massively parallel nascent RNA sequencing methods were established in eukaryotic cells. Here, we systematically compared 3 classes of methods on the same pure or crude nuclei preparations: GRO-seq for sequence nuclear run-on RNAs, pNET-seq for sequence RNA polymerase II-associated RNAs, and CB RNA-seq for sequence chromatin-bound (CB) RNAs in Arabidopsis (Arabidopsis thaliana). To improve the resolution of CB RNAs, 3'CB RNA-seq was established to sequence the 3' ends of CB RNAs. In addition, we modified pNET-seq to establish the Chromatin Native Elongation Transcript sequencing (ChrNET) method using chromatin as the starting material for RNA immunoprecipitation. Reproducibility, sensitivity and accuracy in detecting nascent transcripts, experimental procedures, and costs were analyzed, which revealed the strengths and weaknesses of each method. We found that pNET and GRO methods best detected active RNA polymerase II. CB RNA-seq is a simple and cost-effective alternative for nascent RNA studies, due to its high correlation with pNET-seq and GRO-seq. Compared with pNET, ChrNET has higher specificity for nascent RNA capture and lower sequencing cost. 3'CB is sensitive to transcription-coupled splicing. Using these methods, we identified 1,404 unknown transcripts, 4,482 unannotated splicing events, and 60 potential recursive splicing events. This comprehensive comparison of different nascent/chromatin RNA sequencing methods highlights the strengths of each method and serves as a guide for researchers aiming to select a method that best meets their study goals.

Heterologous expression and characterization of salt-tolerant β-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-β-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-β-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-β-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45°C, respectively. rBGL_MK86 acted on the β-1,3-linked glucose dimer laminaribiose, but not β-1,4-linked or β-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of β-1,3 glucan) from Laminaria digitata than laminarin (β-1,3/β-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on β-1,6-linked glucose residues. The β-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.