Impact of protectants and the method of preservation on the stability of potentially probiotic bacteria.

Probiotics offer health advantages when consumed in adequate quantities. As ongoing research identifies promising new strains, ensuring their viability and functionality through simple preservation methods is vital for success within the probiotic industry. This study employed a factorial design to investigate the combined effects of four cryoprotectants [C1: MRS broth + 14 % (w/v) glycerol, C2: Aqueous solution containing 4 % (w/v) trehalose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate, C3: Aqueous solution containing 10 % (w/v) skimmed milk and 4 % (w/v) sodium glutamate, C4: Aqueous solution containing 4 % (w/v) sucrose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate] and three methods of preservation (P1: -86 °C freezing, P2: -196 °C liquid nitrogen freezing, and P3: storing at 4 °C after lyophilization) on the cell viability of three potentially probiotic strains over 12 months. Pediococcus sp P15 and Weissella cibaria ml6 had the highest viability under treatments C3 and C2, after 12 months of storage, respectively. Meanwhile, Lactococcus lactis ml3 demonstrated the highest viability in both treatments C2 and C4 (P ≤ 0.05). According to the results freezing, either P1 or P2, is the most effective preservation method for P. sp P15 and W. cibaria ml6. Meanwhile, L. lactis ml3 showed the highest colony count under treatment (P1) after 12 months of storage (P ≤ 0.05). Among the tested conditions, P. sp P15 and L. lactis ml3 exhibited the highest viability and bile salt resistance when stored under P1C1. For W. cibaria ml6, the optimal storage condition was P2C2 (frozen in liquid nitrogen with cryoprotectant C2).

A Fingertip-Mimicking 12×16 200µm-Resolution e-skin Taxel Readout Chip with per-Taxel Spiking Readout and Embedded Receptive Field Processing.

This paper presents an electronic skin (e-skin) taxel array readout chip in 0.18µm CMOS technology, achieving the highest reported spatial resolution of 200µm, comparable to human fingertips. A key innovation is the integration on chip of a 12×16 polyvinylidene fluoride (PVDF)-based piezoelectric sensor array with per-taxel signal conditioning frontend and spiking readout combined with local embedded neuromorphic first-order processing through Complex Receptive Fields (CRFs). Experimental results show that Spiking Neural Network (SNN)-based classification of the chip's spatiotemporal spiking output for input tactile stimuli such as texture and flutter frequency achieves excellent accuracies up to 97.1% and 99.2%, respectively. SNN-based classification of the indentation period applied to the on-chip PVDF sensors achieved 95.5% classification accuracy, despite using only a small 256-neuron SNN classifier, a low equivalent spike encoding resolution of 3-5 bits, and a sub-Nyquist 2.2kevent/s population spiking rate, a state-of-the-art power consumption of 12.33nW per-taxel, and 75µW-5mW for the entire chip is obtained. Finally, a comparison of the texture classification accuracies between two on-chip spike encoder outputs shows that the proposed neuromorphic level-crossing sampling (NLCS) architecture with a decaying threshold outperforms the conventional bipolar level-crossing sampling (LCS) architecture with fixed threshold.

Conjugated Microporous Polymers for Catalytic CO2 Conversion.

Rising carbon dioxide (CO2) levels in the atmosphere are recognized as a threat to atmospheric stability and life. Although this greenhouse gas is being produced on a large scale, there are solutions to reduction and indeed utilization of the gas. Many of these solutions involve costly or unstable technologies, such as air-sensitive metal-organic frameworks (MOFs) for CO2 capture or "non-green" systems such as amine scrubbing. Conjugated microporous polymers (CMPs) represent a simpler, cheaper, and greener solution to CO2 capture and utilization. They are often easy to synthesize at scale (a one pot reaction in many cases), chemically and thermally stable (especially in comparison with their MOF and covalent organic framework (COF) counterparts, owing to their amorphous nature), and, as a result, cheap to manufacture. Furthermore, their large surface areas, tunable porous frameworks and chemical structures mean they are reported as highly efficient CO2 capture motifs. In addition, they provide a dual pathway to utilize captured CO2 via chemical conversion or electrochemical reduction into industrially valuable products. Recent studies show that all these attractive properties can be realized in metal-free CMPs, presenting a truly green option. The promising results in these two fields of CMP applications are reviewed and explored here.

Isolation and characterization of novel Bacillus strains with superior probiotic potential: comparative analysis and safety evaluation.

Despite the current use of some Bacillus spp. as probiotics, looking for and introducing new efficient and safe potential probiotic strains is one of the most important topics in both microbiology and food industry. This study aimed to isolate, identify, and evaluate the probiotic characteristics and safety of some Bacillus spp. from natural sources. Thirty-six spore-forming, Gram-positive, and catalase-positive Bacillus isolates were identified in 54 samples of soil, feces and dairy products. Bacterial identification was performed using 16S rDNA sequencing. To evaluate the probiotic potential of isolates, the resistance of bacterial cells to simulated gastrointestinal tract (GIT) conditions, the presence of enterotoxin genes, their susceptibility to antibiotics, antimicrobial and hemolytic activities and biochemical profiles were investigated. The results revealed that eight sporulating Bacillus spp. isolates fulfilled all tested probiotic criteria. They showed a high growth rate, non-hemolytic and lecithinase activity, and resistance to simulated GIT conditions. These strains exhibited broad-spectrum antibacterial activity against pathogenic bacteria. In addition, they did not exhibit antibacterial resistance to the 12 tested antibiotics. The results of this study suggest that these isolates can be considered as candidates for functional foods and as safe additives to improve diet quality.

In Silico and Experimental Studies on the Effect of α3 and α5 Deletion on the Biochemical Properties of Bacillus thermocatenulatus Lipase.

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively.

Improving the Accuracy of Spiking Neural Networks for Radar Gesture Recognition Through Preprocessing.

Event-based neural networks are currently being explored as efficient solutions for performing AI tasks at the extreme edge. To fully exploit their potential, event-based neural networks coupled to adequate preprocessing must be investigated. Within this context, we demonstrate a 4-b-weight spiking neural network (SNN) for radar gesture recognition, achieving a state-of-the-art 93% accuracy within only four processing time steps while using only one convolutional layer and two fully connected layers. This solution consumes very little energy and area if implemented in event-based hardware, which makes it suited for embedded extreme-edge applications. In addition, we demonstrate the importance of signal preprocessing for achieving this high recognition accuracy in SNNs compared to deep neural networks (DNNs) with the same network topology and training strategy. We show that efficient preprocessing prior to the neural network is drastically more important for SNNs compared to DNNs. We also demonstrate, for the first time, that the preprocessing parameters can affect SNNs and DNNs in antagonistic ways, prohibiting the generalization of conclusions drawn from DNN design to SNNs. We demonstrate our findings by comparing the gesture recognition accuracy achieved with our SNN to a DNN with the same architecture and similar training. Unlike previously proposed neural networks for radar processing, this work enables ultralow-power radar-based gesture recognition for extreme-edge devices.

Identification of a potent dual-function inhibitor for hIMPDH isoforms by computer-aided drug discovery approaches.

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme in de novo biosynthesis of purine nucleotides. Due to this important role, it is a great target to drug discovery for a wide range of activities, especially immunosuppressant in heart and kidney transplantation. Both human IMPDH isoforms are expressed in stimulated lymphocytes. In addition to the side effects of existing drugs, previous studies have mainly focused on the type II isoform. In this study, virtual screening and computer-aided approaches were employed to identify potential drugs with simultaneous inhibitory effects on both human IMPDH isoforms. After Re-docking, Double-step docking, and identification of virtual hits based on the PLANTS scoring function, drug-likeness and ADME-Tox assessments of the topmost ligands were performed. Following further evaluation, the best ligand was selected and, in complex with both isoforms, simulated in monomeric and tetrameric forms using molecular dynamics to evaluate its stability and binding pattern. The results showed a potential drug candidate [(S)-N-(3-hydroxy-1-(4-hydroxyphenyl) propyl)-2-(3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide] with a high inhibitory effect on the two human IMPDH isoforms. This drug-like inhibitor could potentially serve as an immunosuppressant to prevent transplant rejection response by inhibiting B- and T-lymphocyte proliferation. In addition, its effect can be evaluated in various therapeutic targets in which IMPDH is known as a therapeutic target, especially in Covid-19 patients.

Optimization of ultrasound-assisted production of ergosterol from Penicillium brevicompactum by Taguchi statistical method.

Ergosterol as a primary metabolite and precursor of vitamin D2, is the most plentiful mycosterols in fungal cell membrane. Process optimization to increase the yield and productivity of biological products is a topic of interest. Ultrasonic waves have many applications in biotechnology, like cell disruption, and enhancement of primary and secondary metabolites production. This study disclosed an optimal condition for ultrasound-assisted production (UAP) of ergosterol from Penicillium brevicompactum MUCL 19,011 using L9 Taguchi statistical method. The intensity (IS), time of sonication (TS), treatment frequency (TF), and number of days of treatment (DT) were allocated to study the effects of ultrasound on ergosterol production. The results were analyzed using Minitab version 19. The maximum ergosterol, 11 mg/g cell dry weight (CDW), was produced on the tenth day while all factors were at a low level. The days of treatment with a contribution of 45.48% was the most significant factor for ergosterol production. For the first time, this study revealed the positive effect of ultrasound on the production of ergosterol. Ergosterol production increased 73% (4.63 mg/g CDW) after process optimization. Finally, a mathematical model of ultrasound factors with a regression coefficient of R2 = 0.978 was obtained for the ergosterol production during ultrasound treatment.

Determinants of pyelonephritis onset in patients with obstructive urolithiasis.

BACKGROUND: Acute obstructive pyelonephritis due to urolithiasis represents a medico-surgical emergency that can lead to life-threatening complications. There are still no established factors that reliably predict progression toward acute pyelonephritis in patients presenting with a simple renal colic. OBJECTIVE: To investigate clinical and paraclinical factors that are associated with the onset of acute obstructive pyelonephritis. METHODS: Patients presenting to the emergency department for renal colic with obstructive urolithiasis on imaging were enrolled in the study. Demographic data, vital signs, medical comorbidities, blood test results, urinalysis, and radiological findings were recorded. Obstructive pyelonephritis was defined by the presence of two or more of the following criteria: fever, flank pain or costovertebral angle tenderness, and a positive urine culture. RESULTS: Seventeen patients out of 120 presenting with renal colic, were diagnosed with acute obstructive pyelonephritis (14%). Parameters that were associated with the onset of obstructive pyelonephritis were: diabetes (p = 0.03), elevated CRP (p = 0.01), stone size (>5 mm) (p = 0.03), dilatation of renal pelvis (p = 0.01), peri-renal fat stranding (p = 0.02), and positive nitrites on urinalysis (p < 0.01). Hyperleukocytosis, acute kidney injury, multiple stones, pyuria (>10/mm3), hypertension, and were not associated with the onset of obstructive pyelonephritis. CONCLUSION: This study showed that known diabetic status, elevated CRP, positive urine nitrites, stone size (>5 mm), pyelic dilatation, and peri-renal fat stranding were associated with the onset of pyelonephritis in patients presenting to the emergency department with obstructive urolithiasis.

Production of Mycophenolic Acid by a Newly Isolated Indigenous Penicillium glabrum.

Soil-occupant fungi produce a variety of mycotoxins as secondary metabolites, one of which is mycophenolic acid (MPA), an antibiotic and immunosuppressive agent. MPA is mainly produced by several species of Penicillium, especially Penicillium brevicompactum. Here, we present the first report of MPA production by a local strain belonging to Penicillium glabrum species. We screened ascomycete cultures isolated from moldy food and fruits, as well as soils, collected from different parts of Iran. MPA production of one hundred and forty Penicillium isolates was analyzed using HPLC. Three MPA producer isolates were identified, among which the most producer was subjected to further characterization, based on morphological and microscopic analysis, as well as molecular approach (ITS, rDNA and beta-tubulin gene sequences). The results revealed that the best MPA producer belongs to P. glabrum IBRC-M 30518, and can produce 1079 mg/L MPA in Czapek-Dox medium.

Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacterxylinus in E. coli Nissle 1917.

Based on cellulose biosynthesis pathway of Gluconacetobacterxylinus BPR2001 and E. coli Nissle 1917, bcsA and bcsB genes have been selected and bioinformatics studies done to the analyses of nucleotide and amino acid sequence alignment, stability of RNA, protein, and promotor power. We amplify and clone bcsA, bcsB, and bcsAB genes of G. xylinus BPR2001 in Escherichiacoli Nissle 1917 under the inducible tac promoter. Our results of bioinformatics predictions demonstrate similar active site and three-dimensional structure of BcsA and BcsB proteins in two different bacteria. In addition, our data reveal that BcsA and BcsB proteins of E. coli have weaker promotor power, RNA secondary structure, and protein stability than that of the same proteins in G. xylinus. Some of the reasons of BcsAB protein selection from G. xylinus and its heterologous expression in E. coli is the noted points. Production of the related proteins visualized using SDS-PAGE. We find out that Congo red absorbance at 490 nm has no significant difference in wild-type strain (E. coli Nissle 1917) compared to recombinants bcsA+ or bcsB+, but recombinant bcsAB+ could produce more cellulose than that of the wild-type strain. Furthermore, the measurement of cellulose dry weights of all samples confirms bacterial cellulose production enhancement in recombinant bcsAB+ (1.94 g l-1). The FTIR analysis reveals that the crystallinity indices do not change significantly after over expressing each of genes.

Determination of oxidative stress levels and some antioxidant enzyme activities in prostate cancer.

In this study, the antioxidant enzyme activities such as (SOD, GSH, and CAT) and malondialdehyde (MDA) level which is the end product of lipid peroxidation, were determined from the serum samples taken from patients diagnosed with prostate cancer Van Yuzuncu Yil University Medical Faculty of Educational Research and Training Hospital and Istanbul Bagcilar Education Research Hospital. The SOD, GSH, and CAT activity of patient groups was found significantly lower than the healthy control group in patients with prostate cancer (p < .05). Serum MDA level is found significantly high when compared to control groups. MDA levels increased in patients that suffer prostate cancer disorder. Whereas, firstly antioxidant enzymes activity of SOD, GSH and CAT have been decreased in control groups. Thus, we concluded that the cause of development of prostate cancer may be the result of an imbalance between the antioxidants and oxidative stress. As a result, SOD, CAT, GSH, and MDA may play an important role in the etiopathogenesis of prostate cancer.

Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

OBJECTIVES: To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. RESULTS: The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. CONCLUSION: The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

Characterization of a farnesyl diphosphate synthase gene from Penicillium brevicompactum MUCL 19011.

OBJECTIVES: Farnesyl diphosphate synthase is a critical enzyme in the isoprenoids biosynthesis pathway responsible for ergosterol and secondary metabolites biosynthesis in fungi. RESULTS: Characterization of fds from Penicillium brevicompactum (Pbfds) was performed using TAIL-PCR and RT-PCR followed by complementation tests in Saccharomyces cerevisiae and determination of its expression profile by semi-quantitative RT-PCR. Promoter analysis suggests some binding sites for transcription factors some of which are involved in fungal growth and response to environmental stress. The Pbfds ORF encodes a cytosolic 39.7 kDa protein with a high conservation among Eurotiomycetes and the highest identity (96 %) with Pen. chrysogenum. Homology-based structural modeling suggests that the PbFDS is formed by the arrangement of 15 core helices around a large central cavity where the catalytic reaction takes place. Superimposition of the predicted 3D structure of the enzyme on its ortholog in human reveals the same folding pattern in the counterparts. CONCLUSION: The Pbfds expression may be stimulated in response to the environmental stresses and fungal growth and encodes the PBFDS--a cytosolic enzyme which with a key role in ergosterol and secondary metabolites biosynthesis.

High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli.

The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1 mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486 mg l(-1), at 3 h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.

Optimization of the BoNT/A-Hc expression in recombinant Escherichia coli using the Taguchi statistical method.

The use of the recombinant BoNT/A-Hc (carboxylic domain of the Clostridium botulinum neurotoxin heavy chain) has been proposed as a vaccine candidate for botulism. This fragment contains the principle protective antigenic determinant. In the present study, in order to maximize recombinant protein expression, after verification of recombinant BoNT/A-Hc by Western blotting, modified M9 medium was selected as a simple medium, and the operational and medium-composition variables together with their interactions were optimized by using the Taguchi statistical method. ANOVA for the obtained data indicated that 3.5 g, 15 g, 30 g, 15 g, 4 g, 0.7 mM, 1.5 ml per litre of medium, 30 degrees C and 15 h represented optimum values of (NH(4))(2)SO(4), glucose, K(2)HPO(4), KH(2)PO(4), MgSO(4) * 7H(2)O, isopropyl beta-D-thiogalactoside concentration, trace-elements solution, temperature and post-induction time respectively. Consequently, under these optimum conditions, 52.1 mg/l of soluble BoNT/A-Hc was obtained in shake flask culture.

Determination of celecoxib in human plasma by high-performance liquid chromatography.

A high performance liquid chromatographic method for the quantitation of celecoxib (CEL) in human plasma is presented. The method is based on liquid-liquid extraction with chloroform and reversed-phase chromatography using a Nucleosil CN column (250 mm x 4.6 mm i.d., 5 microm particle size) and UV spectrophotometer detection at 260 nm. The mobile phase consists of acetonitrile:water (60:40 (v/v)). Flutamide was used as internal standard (IS). The assay was linear in the concentration range of 10-1000 ng/ml when 0.5 ml aliquots of plasma were extracted. Within-day and between-day precision expressed by relative standard deviation is less than 4% and inaccuracy does not exceed 3%. The assay was used to analyze samples collected during human clinical studies.

Selection of a suitable strain from recombinant Escherichia coli strains with the same genetic structure expressing periplasmic hGM-CSF.

The selection of a suitable strain among five recombinant Escherichia coli strains with the same genetic structure that expresses the human granulocyte macrophage colony stimulating factor (hGM-CSF) was carried out based on four criteria:growth rate, expression level, plasmid stability and feasibility for protein extraction. There was no significant difference in growth between the five strains, while a suitable expression level, a high plasmid stability and a good feasibility for protein extraction from periplasmic space were observed for one of the recombinant strains. This strain expressed 27% hGM-CSF relative to total proteins and had 96% plasmid stability after 7-d subcultures on an antibiotic-free LB medium.