Type 2 diabetes mellitus (T2D) is a metabolic disorder characterized by inappropriate insulin function. Despite wide progress in genome studies, defects in gene expression for diabetes prognosis still incompletely identified. Prolonged hyperglycemia activates NF-κB, which is a main player in vascular dysfunctions of diabetes. Activated NF-κB, triggers expression of various genes that promote inflammation and cell adhesion process. Alteration of pro-inflammatory and profibrotic gene expression contribute to the irreversible functional and structural changes in the kidney resulting in diabetic nephropathy (DN). To identify the effect of some important NF-κB related genes on mediation of DN progression, we divided our candidate genes on the basis of their function exerted in bloodstream into three categories (Proinflammatory; NF-κB, IL-1B, IL-6, TNF-α and VEGF); (Profibrotic; FN, ICAM-1, VCAM-1) and (Proliferative; MAPK-1 and EGF). We analyzed their expression profile in leukocytes of patients and explored their correlation to diabetic kidney injury features. Our data revealed the overexpression of both proinflammatory and profibrotic genes in DN group when compared to T2D group and were associated positively with each other in DN group indicating their possible role in DN progression. In DN patients, increased expression of proinflammatory genes correlated positively with glycemic control and inflammatory markers indicating their role in DN progression. Our data revealed that the persistent activation NF-κB and its related genes observed in hyperglycemia might contribute to DN progression and might be a good diagnostic and therapeutic target for DN progression. Large-scale studies are needed to evaluate the potential of these molecules to serve as disease biomarkers.

The seeds of cultivated peanut, Arachis hypogaea, are an agronomically important crop produced for human nutrition, oilseed and feed stock. Peanut seed is the single most expensive variable input cost and thus producers require seed with excellence performance in terms of germination efficiency. During the maturation process, triglycerides are stored in oil bodies as an energy resource during germination and seedling development. The stability of oil body membranes is essential for nutrient mobilization during germination. This study focused on evaluating the phytosterol composition in seed components including the kernel, embryo (heart), and seed coat or skin. Samples of different maturity classes were analyzed for macronutrient and phytosterol content. The three most abundant phytosterols, β-sitosterol, campesterol, and stigmasterol, comprised 82.29%, 86.39%, and 94.25% of seed hearts, kernels, and seed coats, respectively. Stigmasterol concentration was highest in the seed kernel providing an excellent source of this sterol known to have beneficial effects on human health. Peanut hearts contained the highest concentration of sterols by mass potentially providing protection and resources for the developing seedling. The amount of α-tocopherol increases in peanut hearts during the maturation process providing protection from temperature stress and stability required for seedling vigor. These results suggest that phytosterols may play a significant role in the performance of seeds and provides a possible explanation for the poor germination efficiency of immature seeds.

For the first time, We do fuzzification the concept of domination in crisp graph on a generalization of fuzzy graph by using membership values of vertices, α-strong edges and edges. In this paper, we introduce the first variation on the domination theme which we call vertex domination. We determine the vertex domination number γ_v for several classes of t-norm fuzzy graphs which include complete t-norm fuzzy graph, complete bipartite t-norm fuzzy graph, star t-norm fuzzy graph and empty t-norm fuzzy graph. The relationship between effective edges and α-strong edges is obtained. Finally, we discuss about vertex dominating set of a fuzzy tree with respect to a t-norm ⨂ by using the bridges and α-strong edges equivalence.

The mechanistic pathway of TEMPO/I₂-mediated oxidative cyclization of N,N-diaryl amino alcohols 1 is investigated in this study. Based on our direct empirical experiments, three key intermediates (the aminium radical cation 3, the α-aminoalkyl radical 4, and the iminium 5), four kind reactive species (radical TEMPO, cationic TEMPO, TEMPO-I and iodo radical) and three kind pathways (1. SET/PCET mechanism, 2. HAT/1,6-H transfer mechanism, 3. Ionic mechanism) were assumed. Under the assumption, nine free energy diagrams are acquired through DFT calculation. From the comparison of the solution phase free energy, some possibility can be excluded and then the chosen plausible mechanisms are concretized with the more stable intermediate 7.

Aggregated alpha-synuclein (-synuclein) is the main component of Lewy bodies (LBs), Lewy neurites (LNs), and glial cytoplasmic inclusions (GCIs), which are pathological hallmarks of idiopathic Parkinson’s disease (IPD) and multiple system atrophy (MSA), respectively. Initiating factors that culminate in forming LBs/LNs/GCIs remain elusive. Several species of -synuclein exist, including phosphorylated and nitrated forms. It is unclear which -synuclein post-translational modifications (PTMs) appear within aggregates throughout disease pathology. Herein we aimed to establish the predominant synuclein PTMs in post-mortem IPD and MSA pathology using immunohistochemistry. We examined the patterns of three -synuclein PTMs (pS87, pS129, nY39) simultaneously in pathology-affected regions of 15 PD, 5 MSA, 6 neurologically normal controls. All antibodies recognized LBs, LNs, and GCIs, albeit to a variable extent. pS129 -synuclein antibody was particularly immunopositive for LNs and synaptic dot-like structures followed by nY39 -synuclein antibody. GCIs, neuronal inclusions, and small threads were positive for nY39 -synuclein in MSA. Quantification of the LB scores revealed that pS129 -synuclein was the dominant and earliest -synuclein PTM followed by nY39 -synuclein, while lower amounts of pSer87 -synuclein appeared later in disease progression in PD. These results may have implications for novel biomarker and therapeutic developments.

The presence of valuable bioactive compounds in European eel (Anguilla anguilla) skin was studied. For it, proximate and lipid class compositions and analysis of the fatty acid (FA) profile (individual FAs; FA groups, i.e., saturated, monounsaturated, and polyunsaturated; FA ratios, i.e., polyunsaturated/saturated, ω3/ω6) were determined and compared to the composition of the eel muscle. As a result, higher (p<0.05) levels of proteins (271.6 g·kg-1), lipids (38.0 g·kg-1), ash (27.7 g·kg-1) and ω6 FAs were observed in the skin tissue. Contrary, the muscle tissue showed higher (p<0.05) moisture, ω3 FA and ω3/ω6 values. Regarding lipid classes, a higher (p<0.05) proportion of phospholipids (111.1 g·kg-1 lipids), sterols (104.7 g·kg-1 lipids), α-tocopherol (274.0 mg·kg-1 lipids), and free fatty acids (43.6 g·kg-1 lipids) was observed in the skin tissue. No differences (p>0.05) between both tissues could be detected for triacylglycerol and FA group (saturated, monounsaturated, and polyunsaturated) values, as well as for the polyunsaturated/saturated ratio. It is concluded that eel skin, a by-product resulting from commercial processing, can be considered a valuable source to provide the food and pharmaceutical industries with useful value-added constituents such as proteins, lipids, ω3 FAs, phospholipids, and α-tocopherol.

In this study, we aimed to reveal the proinflammatory effects of serum 25-hydroxyvitamin D3 (Vit D) deficiency and insufficiency in new-onset type 2 diabetes mellitus (T2DM) and prediabe-tes. We recruited 84 prediabetes patients, 94 new-onset T2DM patients and 113 healthy partici-pants. Levels of C-reactive protein (CRP), fibrinogen, ferritin, interleukin-1β (IL-1β), interleu-kin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) were measured in the serum of the par-ticipants. The ANOVA Bonferonni and Kruskal Wallis Dunn tests were used for the comparison of the inflammation markers and vitamin D levels between the groups. Upon covariance analy-sis with age, gender and BMI; Vit D levels of T2DM group were found to be significantly lower (p<0.003). Proinflammatory markers and CRP were found to be significantly higher in predia-betic and diabetic subjects. In the prediabetes group, IL-1β, IL-6, IL-8, TNF-α and MAPK were significantly higher in those with Vit D insufficiency and deficiency groups. In T2DM group, IL-1β, IL-6, IL-8, TNF-α, NF-κB, MAPK and CRP were significantly higher in those with Vit D insufficiency and deficiency. Our study emphasizes the proinflammatory effects of Vit D on the initiation of inflammation in both prediabetes and diabetes mellitus.

The Golgi apparatus (GA) dysfunctions in Parkinson’s Disease (PD), neurodevelopmental disorders (NDDs), cancer, and organelle structural biology (OSB) can provide insights into therapeutic targets, gene therapy, and drug design. Primary defects and fragmentation within the GA are implicated in a wide range of neurodegenerative diseases. GA defects typically result in mislocation of proteins, accumulation of undegraded proteins, and impaired glycosylation of proteins. Inhibition of vesicular trafficking by α-synuclein (aSyn) may affect the dopamine-producing neurons and neuromodulators. GA regulates apoptosis during pathological mechanisms of neurological diseases and could provide new avenues in treatments through translation research. PD patients bearing the hereditary E46K disease mutation manifest the clinical picture of parkinsonism. How do we provide high resolution nanoimages of the GA during disease to capture dysfunction? Could we visualize the aSyn traffic jam between vesicles in the organelles ER and GA? OSB is emerging as a field as more technology advances and is more accessible. Structural studies of the GA will advance the field of neurological disease forward with an in depth understanding of dysfunction, fragmentation, and defects. Discoveries of the GA in PD, NDDs, and cancer would break new ground and provide translational medicine data of these diseases. Future research could be visualizing high angle annular dark field-STEM (HAADF-STEM) tomograms, cryogenic electron tomography (cryo-ET), multiplex correlative light and electron microscopy (cryo-CLEM), nanobody-assisted tissue immunostaining for volumetric EM (NATIVE) and using soft X-ray tomography (SXT) and computational reconstruction of the GA.

Zinc, being a part of many proteins and enzymes, plays an important role in the vital processes of the body. Racemic α-lipoic acid (LA) has a wide range of biological activities: antioxidant, antidiabetic, anticancer, and it is also used in diseases such as Parkinson's and Alzheimer's. Nowadays, the synthesis and structural studies of new zinc complexes with biologically active compounds are of great importance for drug discovery. This work shows the studies carried out on the synthesis, structure, identification, also biological activity using in silico methods of a complex compound based on LA and zinc. A complex compound of zinc with LA (ZnLA) was synthesized in the presence of LA and zinc acetate in ethanol. The identification of ZnLA by (FT-IR, XRD and TGA-DTA), also DFT calculations at the B3LYP/def2-TZVPP level of theory was performed. The molecular docking investigation involved docking ZnLA with two different protein receptors such us Glycogen phosphorylase (2GPA) and Kv potassium channel beta subunit (1ZSX) using CB-Dock 2 server and Auto-Dock Vina program to determine the mechanism of action and biological activity. This study reveals potential ligand-protein interactions at the atomic level and suggests that the treatment of diabetes (non-insulin dependent) with ZnLA.

Beef meat production in Uruguay is based on pasture or concentrate feeding systems, leading to differences in meat quality. The objective of this study was to compare the oxidative and color stability during refrigerated retail display of beef meat from these two systems. For this, ten Aberdeen Angus steers were reared and fed on pasture and other ten were finished on a concentrate diet. After slaughter, the Semimembranosus muscle was cut into slices and put in a refrigerated showcase for 3, 6, and 9 days simulating commercial conditions. The β-carotene, α-tocopherol, and fatty acid content were determined before the display, and the lipid and protein oxidation, color parameters, and heme iron content were determined before and during the display. Meat from steers fed pasture had a lower content of intramuscular fat, monounsaturated fatty acids, n-6/n-3 ratio, lipid and protein oxidation, L* and a* values, and a higher content of linolenic acid, DHA, total n-3, β-carotene, and α-tocopherol. In conclusion, meat from pasture-fed steers was more stable during the retail display, which can probably be explained by a higher content of antioxidant compounds, such as β-carotene and α-tocopherol, and presented a healthier fatty acid profile.

To enhance the mechanical properties (i.e. strength and elongation) of the face-centered cubic (fcc) α-phase in the Au-Cu-Al system, this study focused on the introduction of the martensite phase (doubled B19 (DB19) crystal structure of Au2CuAl) via the manipulation of alloy compositions. Fundamental evaluations, such as microstructure observations, phase identifications, thermal analysis, tensile behavior examinations, and reflectance analysis have been conducted. The presence of fcc annealing twins was both observed in the optical microscope (OM) and the scanning electron microscope (SEM) images. Both the strength and elongation of the alloys were greatly promoted while the DB19 martensite phase was introduced into the alloys. Amongst all the prepared specimens, the 47Au41Cu12Al and the 44Au44Cu12Al alloys performed the optimized mechanical properties. The enhancement of strength and ductility in these 2 alloys was achieved while the stress plateau was observed during the tensile deformation. A plot of the ultimate tensile strength (UTS) against fracture strain was constructed to illustrate the effects of the introduction of the DB19 martensite phase on the mechanical properties of the alloys. Regardless of the manipulation of the alloy compositions and the introduction of the DB19 martensite phase, the reflectance stayed almost identical to pure Au.

We report the results of our study of approved drugs as potential treatments for COVID 19, based on the application of various bioinformatics predictive methods. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). Our results indicate that these small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.

Background: Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here we report studies that address this question. Methods: 14 weeks on high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Results: Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and prevent cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Conclusions: Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

Obesity and metabolic syndrome involve chronic low-grade inflammation called metabolic inflammation as well as metabolic derangements from increased endotoxin and free fatty acids. It is debated whether the endoplasmic reticulum (ER) stress in monocytic cells can contribute to amplify metabolic inflammation; if so, by which mechanism(s). To test this, metabolic stress was induced in THP-1 monocytic cells by treatments with lipopolysaccharide (LPS), palmitic acid (PA), or oleic acid (OA), in the presence or absence of ER stressor thapsigargin (TG). Gene expression of tumor necrosis factor (TNF)-α and markers of ER/oxidative stress was determined by qRT-PCR, TNF-α protein by ELISA, ROS by DCFH-DA assay, HIF-1α/p38/ERK-1,2/NF-κB phosphorylation by immunoblotting, and insulin sensitivity by glucose-uptake assay. Regarding clinical analyses, adipose TNF-α was assessed by qRT-PCR/IHC and plasma TNF-α/hs-CRP/MDA/OX-LDL by ELISA. We found that the cooperative interaction between metabolic and ER stresses promoted TNF-α, ROS, CHOP, ATF6, SOD2, and NRF2 expression (P≤0.0183),. However, glucose uptake was not impaired. TNF-α amplification was dependent on HIF-1α/p38/NF-κB phosphorylation, while MAPK/NF-κB inhibitors and antioxidants/ROS scavengers attenuated TNF-α production (P≤0.05). Individuals with obesity displayed increased adipose TNF-α gene/protein expression as well as elevated plasma levels of TNF-α, CRP, MDA, and OX-LDL (P≤0.05). Our findings support a cooperative interaction between metabolic and ER stresses, favoring inflammation by triggering TNF-α production via the ROS/CHOP/HIF-1α and MAPK/NF-κB dependent mechanisms. This study also shows the therapeutic potential of ROS scavengers/anti-oxidants in inflammatory conditions involving metabolic/ER stresses.

Chlorotoxin (CTX) is a 36–amino acid peptide with 8 Cys residues that forms four Cys-Cys bonds. It has high affinity for the glioma-specific chloride channel and matrix metalloprotease-2. Structural and binding properties of CTX analogs with various Cys residue substitutions with L-a-aminobutyric acid (Abu) have been previously reported. Using 4.2 ìs molecular dynamics, we compared the conformational and essential space sampling of CTX and analogs [Abu/Ser^2,19]CTX, [Abu/Ser^5,28]CTX, [Abu/Ser^16,33]CTX, [Abu/Ser^20,35]CTX, and [Abu/Ser^{2,5,16,19,20,28,33,35}]CTX. The native and substituted peptides maintained a high degree of a-helix propensity from residues 8 through 21, with the exception of [Ser^5,28]CTX and [Abu^16,33]CTX. In agreement with previous circular dichroism spectropolarimetry results, the C-terminal b-sheet content varied less from residues 25 through 29 and 32 through 36 and was well conserved in all analogs, except: [Abu^16,33]CTX, [Ser^20,35]CTX, [Abu^{2,5,16,19,20,28,33,35}]CTX, and [Ser^{2,5,16,19,20,28,33,35}]CTX. The Cys¹⁶-Cys³³ and Cys²⁰-Cys³⁵ Cys-Cys bonds appear to be required to maintain the ab-motif of CTX. Their selective substitution with Ser^16,33 however, may mitigate the destabilizing effect of Cys¹⁶-Cys³³ substitution by the formation of an inter residue H-bond from OgH of Ser¹⁶ to OgH of Ser³³ bridged by a water molecule. All peptides shared considerable sampled conformational space, which explains the retained receptor binding of the nonnative analogs.

Acetic acid bacteria (AAB) form a bacterial film on the surface of alcoholic solutions and ferment ethanol to acetic acid while also producing bioactive compounds. To discover functional AAB for industrial use, we isolated and selected strains from farm-produced vinegars using a CaCO3-containing medium. The seven strains belonged to Acetobacter cerevisiae and A. pasteurianus. Physiological properties were determined, including ethanol tolerance at up to 12% (v/v). Acidification at a growth temperature of 40 ℃ was seen for GHA 7, GYA 23, JGB 21-17, and GHA 20 strains, which may be useful in vinegar production where thermotolerant AAB are required. The seven AAB isolates had strong antibacterial activity against Staphylococcus aureus. Antioxidant activity was 2- and 4-fold higher, respectively, for A cerevisiae and A. pasteurianus than for the control, 1% acetic acid. We also observed 91.3% inhibition of angiotensin-converting enzyme activity for the KSO 5 strain, higher than that for the positive control, 0.1% captopril (76.9%). All strains showed complete inhibition of α-glucosidase, except JGB 21-17 and GHA 7 with 98.3% inhibition. Our work suggests usefulness of the strains selected as seed strains for highly efficient production of functional vinegar and illustrates identification of useful functional characteristics on a scientific basis.

We address weighted composition operators ψCφ from α-Bloch spaces to Bers-type spaces of bounded holomorphic functions on Y, where Y is a generalized Hua domain of the first kind, and obtain some necessary and sufficient conditions for the boundedness and compactness of those operators.

A Multicomponent Reaction (MCR) strategy, alternative to the known cycloaddition reaction, towards variously substituted 1-amino-1H-imidazole-2(3H)-thione derivatives has been successfully developed. The novel approach involves α-halohydrazones whose azidation process followed by tandem Staudinger/aza-Wittig reaction with CS₂ in a sequential MCR regioselectively leads to the target compounds avoiding the formation of the regioisomer iminothiazoline heterocycle. The approach can be applied to a range of differently substituted α-halohydrazones bearing also electron-withdrawing groups confirming the wide scope and the substituent tolerance of the process for the synthesis of the target compounds. Interestingly, the concurrent presence of reactive functionalities in the scaffolds so obtained, ensures postmodifications in view of N-bridgedheaded heterobicyclic structures.

Alzheimer’s disease manifests itself as a complex pathological condition with neuroinflammation, oxidative stress and cholinergic dysfunction being a few of the many pathological changes. Due to the complexity of the disease current therapeutic strategies aim at a multitargeted approach often relying on a combination of sub-stances with versatile and complementary effects. In the present study, unique combination of α-lipoic acid, citicoline, extracts of leaves from olive tree and green tea, Vitamin D3, selenium and an immune supporting complex was tested in a scopolamine-induced dementia in rats. Using behavioral and biochemical methods we assessed the effects of this combination on learning and memory, and elucidated the mechanisms under-lying its effects. Our results showed that as compared to the components, experimental combination was most efficient in improving short- and long-term memory assessed by the step-through method as well as spatial memory, assessed by T-maze and Barnes maze underlying decrease in AChE activity and LPO, in-creases in SOD activity in cortex; activities of catalase and GPx, levels of BDNF and pCREB in the hippocam-pus. No significant histopathological changes or blood parameter changes were detected, making the experi-mental combination an effective and safe candidate in a multitargeted treatment of AD.

Cannabinoids, mainly cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), are the most studied group of compounds obtained from Cannabis sativa because of their several pharmaceutical properties. Current evidence suggests a crucial role of cannabinoids as potent anti-inflammatory agents for the treatment of chronic inflammatory diseases; however, the mechanisms remain largely unclear. Cytokine storm, a dysregulated severe inflammatory response by our immune system, is involved in the pathogenesis of numerous chronic inflammatory disorders, including coronavirus disease 2019 (COVID-19), which results in the accumulation of pro-inflammatory cytokines. Therefore, we hypothesized that CBD and THC reduce the levels of pro-inflammatory cytokines by inhibiting key inflammatory signalling pathways. The nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signalling has been implicated in a variety of chronic inflammatory diseases, which results in the release of pyroptotic cytokines, interleukin-1β (IL-1β) and IL-18. Likewise, the activation of the signal transducer and activator of transcription-3 (STAT3) causes increased expression of pro-inflammatory cytokines. We studied the effects of CBD and THC on lipopolysaccharide (LPS)-induced inflammatory response in human THP-1 macrophages and primary human bronchial epithelial cells (HBECs). Our results revealed that CBD and, for the first time, THC, significantly inhibited NLRP3 inflammasome activation following LPS + ATP stimulation, leading to a reduction in the levels of IL-1β in THP-1 macrophages and HBECs. CBD attenuated the phosphorylation of nuclear factor-κB (NF-κB) and both cannabinoids inhibited the generation of oxidative stress post-LPS. Our multiplex ELISA data revealed that CBD and THC significantly diminished the levels of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) after LPS treatment in THP-1 macrophages and HBECs. In addition, the phosphorylation of STAT3 was significantly downregulated by CBD and THC in THP-1 macrophages and HBECs, which was in turn, attributed to the reduced phosphorylation of tyrosine kinase-2 (TYK2) by CBD and THC after LPS stimulation in these cells. Overall, CBD and THC were found to be effective in alleviating the LPS-induced cytokine storm in human macrophages and primary HBECs, at least via modulation of NLRP3 inflammasome and STAT3 signalling pathways. The encouraging results from this study warrant further investigation of these cannabinoids in vivo.

A metastable β-Ti alloy, Ti–10V–3Fe–3Al (wt.%), was subjected to thermos-mechanical processing including the compression test at 725°C, which is below the β transus temperature (780°C), and at strain rate of 10-3s-1. The presence of phases was determined using transmission electron microscopy and X-ray diffraction. Although the dynamic recovery took place together with slip, both deformation-induced α˝ martensite and ω were detected as other operating mechanisms for the first time in metastable-β Ti alloys deformed in α+β region. The volume fraction of stress-induced α˝ was higher than that of the same alloy deformed at room temperature due to higher strain applied. Stress-induced twinning was not operational, which could be related to the priority of slip mechanism at high temperature resulted from thermally-assisted nucleation and lateral migration of kink-pairs.

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPS clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy. Subsequent transfection with mRNA encoding excision-only transposase allows for the generation of footprint-free isogenic iPSC lines. These monoclonal isogenic iPSC lines retain a normal molecular genotype, express pluripotency markers and have the ability to differentiate into the three germ layers. This combinatory approach of FACS, HCS and post-sorted restriction digestion facilitates the generation of isogenic cell lines for disease modelling to be scaled-up on an automated platform.

Understanding the phenomena underlying the non-selective susceptibility to ischemia of py-ramidal neurons in the CA3 area of the hippocampus is important from the point of view of elu-cidating the mechanisms of memory loss and the development of post-ischemic dementia. We used an ischemic model of Alzheimer's disease to study changes in amyloid protein precursor gene expression, its cleavage enzymes and tau protein in the CA3 area of the hippocampus af-ter a 10-minute brain ischemia with 12, 18, and 24-month survival. Quantitative reverse tran-scriptase PCR assay showed that the expression of all the genes that contribute to amyloid pro-duction was dysregulated within 2 years in the CA3 area of the hippocampus after ischemia. The expression of the amyloid protein precursor gene was above the control values at all times of the study. The expression of the α-secretase gene also exceeded the control values throughout the study. In contrast, the expression of the β-secretase gene reaching its maximum increase 12 months after ischemia, was below control values after 18 months and again above control values after 24 months of survival. Presenilin 1 and 2 gene expression was significantly elevated throughout the follow-up period, with peak expression of both genes occurring 12 months after ischemia. This suggests that the genes studied are involved in the non-amyloidogenic processing of amyloid protein precursor. Also, tau protein gene expression was significantly elevated throughout the observation period, and peak gene expression was present 12 months after is-chemia. Data indicate that an episode of brain ischemia with long-term survival causes damage and death of pyramidal neurons in the CA3 area of the hippocampus in a manner dependent on modified tau protein. Thus defining a new and important mechanism of pyramidal neuronal death in the CA3 area after ischemia. In addition tau protein gene modification after brain is-chemia is useful in identifying ischemic mechanisms occurring in Alzheimer's disease.

Ferrocenium (Fc+) inherits a number of molecular/electronic properties from the neutral counterparts’ ferrocene (Fc) including the high symmetry. Both Fc+ and Fc prefer the eclipsed structure (D5h) over the staggered structure (D5d) by an energy of 0.36 kcal·mol-1. The present study using the recently developed excess orbital energy spectrum (EOES) shows that the open shell Fc+ cation exhibits similar conformer dependent configurational changes to the neutral Fc conformer pair. A further energy decomposition analysis (EDA) discloses that the reasons for the preferred structures are different between Fc+ and Fc. The dominant differentiating energy between the Fc+ conformers is the electrostatic energy (EEstat), whereas in neutral Fc, it is the quantum mechanical Pauli repulsive energy (EPauli). Within the D5h conformer of Fc+, the EOES reveals that the -electrons of Fc+ experience more substantial conformer dependent energy changes than the -electrons (assumed the hole is in a β orbital).

Polyunsaturated fatty acids (PUFAs) and antioxidants are important mediators in the central nervous system (CNS). Lipid derivatives may be used to generate endocannabinoids or prostanoids derived from arachidonic acid, which attenuates excitotoxicity in quadripartite synapses with a focus in astrocytes and microglia; on the other hand, antioxidants, such as glutathione (GSH) and ascorbate, have been shown to signal through transmitter receptors and protect against acute and chronic oxidative stress, modulating the activity of different signaling pathways. Several authors have investigated the role of these nutrients in young and senescent brain, as well as in degenerative conditions such as Alzheimer’s and Parkinson's diseases. Through literature review, we aimed to highlight recent data on the role of fatty acids, antioxidants and physical activity in physiology and in molecular mechanisms of brain senescence. Data indicate the complexity and essentiality of endogenous/dietary antioxidants for maintenance of the redox status and control of neuroglial signaling under stress. Recent studies also indicate that omega-3 and -6 fatty acids act in a competitive manner to generate mediators for energy metabolism, feeding behavior, plasticity and memory mechanisms throughout aging. Finding pharmacological or dietary resources that mitigate or prevent neurodegenerative affections continues to be a great challenge and require additional efforts from researchers, clinicians and nutritionists in the field.

A novel approach for the long-term medical treatment of the inner ear is the diffusion of drugs through the round window membrane from a patient-individualized, drug-eluting implant, which is inserted in the middle ear. In this study, drug-loaded (10 wt% Dexamethasone) guinea pig round window niche implants (GP-RNIs, ~1.30 mm x 0.95 mm x 0.60 mm) were manufactured with high precision via micro injection molding (µIM, Tmold = 160 °C, crosslinking time of 120 s). The medical grade silicone elastomer MED-4244 (NuSil Technology LLC, Radnor, PA, USA) was used. Molds for µIM were 3D printed from a commercially available resin PlasGRAY V2 (Asiga, Alexandria, Australia, TG = 84 °C) via a high-resolution DLP process (xy-resolution of 32 µm, z-resolution of 10 µm, 3D printing time of about 6 h). Drug release, biocompatibility, and bio-efficacy of the GP-RNIs were investigated in vitro. GP-RNIs could be successfully produced. Wear of the molds due to thermal stress was observed. However, the molds are suitable for single use in the µIM process. About 10% of the drug load (8.2 ± 0.6 µg) was released after 6 weeks (medium: isotonic saline). The implants showed high biocompatibility over a time of 28 days (lowest cell viability ~80%). Moreover, we found anti-inflammatory effects over a time of 28 days in a TNF-α-reduction test. These results are promising for the development of long-term drug-releasing implants for human inner ear therapy.

Nonalcoholic fatty liver disease (NAFLD) is becoming the leading cause of hepatocellular carcinoma (HCC), liver-related mortality, and liver transplantation. There is reasonable epidemiological cohort data to recommend surveillance of patients with NAFLD based upon the incidence of HCC. The American Gastroenterology Association (AGA) expert review published in 2020 recommend that NAFLD patients with cirrhosis or advanced fibrosis estimated by non-invasive tests (NITs) should consider HCC surveillance. NITs include fibrosis-4 (FIB-4) index, the enhanced liver fibrosis (ELF) test, FibroScan, and MR elastography. The recommended surveillance modality is abdominal ultrasound (US) given that it is cost effective and noninvasive with good sensitivity. However, US is limited in obese patients and those with NAFLD. In NAFLD patients with a high likelihood of having an inadequate US or if US is attempted but inadequate, CT or MRI may be utilized. The GALAD score, consisting of age, gender, AFP, lens culinaris-agglutinin-reactive fraction of AFP (AFP-L3), and protein induced by vitamin K absence or antagonist-II (PIVKA-II), can help to identify high risk of incident HCC in NAFLD patients. Innovative parameters including Mac-2 binding protein glycated isomer , type IV collagen 7S, free apoptosis inhibitor of macrophage, combination of single nucleoside polymorphisms are expected to be established. Considering a large number of NAFLD population, optimal screening tests must meet several criteria including high sensitivity, cost effectiveness and availability.

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of Endoplasmic Reticulum glycoprotein folding Quality Control (ERQC). In particular, the Arabidopsis thaliana (At) innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC in planta have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the PHI-1 and RET-OX genes in the At ER α-Glu II rsw3 and the At UGGT uggt1-1 mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the rsw3 but not completely abrogated in the uggt1-1 mutant plants, raising the possibility that the latter enzyme is partly dispensable for ERF signalling. In the rsw3 mutant, seedling growth was impaired only by concomitant application of the At ER α-Glu II NB-DNJ inhibitor at concentrations above 500 nM, suggesting residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signalling at the molecular level.

High dietary phosphate intake and poor adherence to phosphate-binding-therapy elevate the risk of hyperphosphatemia in maintenance hemodialysis (HD; MHD) patients. Therefore, chronic kidney disease-related mineral and bone disorder (CKD-MBD) indicators increase; consequently, risks of CKD-MBDs and inflammation are elevated. This double-blind, randomized control trial intervention study was designed to investigate the possibility of reducing blood CKD-MBD indicators and modulating inflammatory indicators by consuming low-phosphate (LP) meals accompanied by a minimum dose of a calcium-based phosphate binder (CaCO3). MHD patients were recruited and randomly assigned to an LP meal group (LP group) or a control group. After initial data collection, blood collection, and dietary counseling, subjects were asked to consume a washout diet for 1 week. During the washout diet period, subjects consumed their usual diet but took 1 tablet of calcium carbonate (1CaCO3) as a phosphate binder with each meal. After the washout diet period, subjects in the LP group and control group respectively consumed LP meals and regular meals twice a day for 1 week. Meat in the LP meals was boiled before the regular cooking process, but meat in control meals was not. All meals were supplied by a central kitchen so that the contents of phosphate and other nutrients could be identified. In total, 40 MHD patients completed the study program. After 1 week of the dietary intervention, the blood Ca x P product and dietary phosphate had significantly decreased in the LP group compared to the control group (p&lt;0.05). The LP group had significantly lower variations in dietary phosphate intake, blood calcium, Ca x P product, and tumor necrosis factor (TNF)-α than the control group by comparing differences between after the dietary intervention and the baseline (△after intervention - baseline, p&lt;0.05). The increase in dietary phosphate intake (△3rd - 2nd dietary phosphate intake) augmented the increase in the TNF-α level by 6.24-fold (odds ratio [95% confidence interval]: 6.24 [1.12~34.92], p&lt;0.05). These results highlighted the conclusion that LP meals accompanied by a minimum dose of CaCO3 downregulated pro-inflammation by reducing CKD-MBD indicators which was triggered by decreasing dietary phosphate intake.

Post-stroke depressive disorders (PSD) and post-stroke cognitive impairments (PCI) are frequent consequences of ischemic stroke (IS). The study was focused on exploring possible associations between relative volumes of cortical and limbic brain structures during the acute period of IS, and changes in biochemical indices of hypothalamic-pituitary-adrenal, sympathoadrenal medullary and inflammatory systems, with the development of PSD or PCI after mild or moderate IS. Patients developing PSD later on had significantly smaller relative volumes of the hippocampus, entorhinal cortex, and temporal pole versus patients without depressive symptoms. PCI development was associated with significantly smaller volumes of temporal pole and supramarginal gyrus versus patients without cognitive changes. Multiple logistic regression analysis showed higher likelihood of developing PSD in patients with smaller temporal pole volume (β0=10.9; β=-4.27; p=0.04) and increased salivary α-amylase activity (β0=-3.55; β=2.68e-05; p=0.02,). PCI likelihood was higher in patients with smaller supramarginal gyrus volume (β0=3.41; β=-0.99; p=0.047), smaller temporal pole volume (β0=3.41; β=-3.12; p=0. 06), and increased hair cortisol concentration at admission (index of accumulated stress load within a month before IS; β0=3.41; β=-0.05; p=0.08). The data support the hypothesis suggesting predisposition to PSD and PCI and multi hit scenarios of their pathogenesis with IS providing a final hit.

A series of novel substituted isoxazolidine derivatives was synthesized through 1,3-dipolar cycloaddition reactions of α-aryl-N-methyl nitrones with various ethylene moieties, resulting in the formation of isoxazolidine derivatives. The structures of the synthesized compounds were characterized using spectroscopic methods. The [3+2] cycloaddition (32CA) reactions of α-aryl-N-methyl nitrones with diverse ethylene substituents were computed employing Density Functional Theory (DFT) at the B3LYP/6–31G(d) level, upgrading bond parameters. An investigation of the electron localization function (ELF) unveiled the zwitterionic nature of α-aryl-N-methyl nitrones, devoid of pseudoradical or carbenoid centers. Utilization of CDFT indices enabled the prediction of global electronic flux from the strongly nucleophilic α-aryl-N-methyl nitrones to the varied electrophilic ethylene functionalities. Molecular docking and molecular dynamics simulations were conducted on the isoxazolidine derivatives against the EGFR receptor (PDB: 4ZAU). In silico assessment of the synthesized isoxazolidines involved molecular docking, wherein the compound with the highest docking score was subjected to molecular dynamics simulations. Compound 11a, out of the thirty isoxazolidine derivatives, displayed the most substantial binding energy, yielding a g score of -6.57 kcal/mol. These observations highlighted the functional role of amide NH2 and NO2 in the binding process with the target protein, influencing its activity. Furthermore, prediction of ADME drug-likeness properties was performed to identify a suitable candidate.

In real-life scenario, there are many mathematical tools to handle the incomplete and imprecise data. One of them is the fuzzy approach. This article aims to contribute to the literature of fuzzy multi-objective dynamic programming issues involving the fuzzy objective functions. The piecewise quadratic fuzzy numbers characterize these fuzzy parameters. Some basic notions in the problem under the α-pareto optimal solution concept is redefined and analyzed to study the stability of the problem. Furthermore, a technique, named as enhancing decomposition approach, is presented for achieving a subset for the parametric space that contains the sameα-pareto optimal solution. For a better understanding and comprehension of the suggested concept, a numerical example is provided.

Background: Vascular inflammation plays a crucial role in peripheral arterial disease (PAD), although the role of the mediators involved has not yet been properly defined. The aim of this work is to investigate gene expression and plasma biomarkers in chronic limb-threating ischemia (CLTI). Methods: Using patients from the GHAS trial, both blood and ischemic muscle samples were obtained to analyze plasma markers and mRNA expression, respectively. Statistical analy-sis was performed by using univariate (Spearman, t-Student, X2) and multivariate (multiple lo-gistic regression) tests. Results: 35 patients were available at baseline (29 for mRNA expression). Baseline characteristics (mean): Age:71.4±12.4 (79.4% male); TNF-α:10.7±4.9; hs-CRP:1.6±2.2; Neutrophil-to-lymphocyte ratio (NLR):3.5±2.8. Plasma TNF-α was found elevated (≥8.1) in 68.6% of patients, while high hs-CRP (≥0.5) in 60.5%. Diabetic patients with high level of inflammation showed significantly higher levels of NOX4 expression at baseline (p=0.0346). Plasma TNF-α had a negative correlation with eNOS expression (-0.5, p=0.015) and hs-CRP with VEGF-A (-0.63, p=0.005). The expression of NOX4 was parallel to that of plasma TNF-α (0.305, p=0.037), especial-ly in DM. Cumulative mortality at 12-month was related to NLR ≥3 (p=0.019) and TNF-α ≥8.1 (p=0.048). The best cut-off point for NLR to predict mortality was 3.4. Conclusions: NOX4 and TNF-α are crucial for the development and complications of lower limb ischemia, especially in DM. hs-CRP could have a negative influence on angiogenesis too. NLR and TNF-α represent suita-ble markers of mortality in CLTI. These results are novel because they connect muscle gene expres-sion and plasma information in patients with advanced PAD, deepening the search of new and ac-curate targets for this condition.

The term ‘Hallmarks of Cancer’ was coined by Hanahan and Weinberg in their influential reviews and they described genome instability as a property of cells enabling cancer development [1, 2]. Accurate DNA replication of genomes is central to diminish genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase, during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair is discussed.

Our previous studies showed that in patients with brain diseases neurotrophic factors in lacrimal fluid (LF) may change more prominently than in blood serum (BS). Since glial cell line-derived neurotrophic factor (GDNF) is involved in control of neuronal networks in epileptic brain, we aimed to assess GDNF levels in LF and BS as well as BDNF, hypothalamic-pituitary-adrenocortical and inflammation indices in BS of patients with focal epilepsy (PWFE), epilepsy and comorbid depression (PWFE+MDD) compared with major depressive disorder (PWMDD) and healthy controls (HC). GDNF levels in BS were similar in patients and HC and higher in PWFE taking valproates. GDNF levels in LF were significantly lower in all patient groups compared to controls, and independent of drugs used. LF and BS GDNF concentrations positively correlated in HC, but not patient groups. BDNF level was lower in BS of patients compared with HC and higher in PWFE+MDD taking valproates. Logistic regression models demonstrated that GDNF decrease in LF may be an important predictor of focal epilepsy. Probability of focal epilepsy can be evaluated using GDNF in LF and BDNF in BS; MDD - GDNF in LF, cortisol and TNF-α in BS; epilepsy with MDD - GDNF in LF, TNF-α and BDNF in BS.

The sexual reproduction of ascomycetes is controlled by the mating-type(MAT) locus. (Pseudo)homothallic reproduction has been hypothesized based on genetic data from Hirsutella sinensis (Genotype #1 of Ophiocordyceps sinensis). In this study, the differential occurrence of the mating-type genes in the MAT1-1 and MAT1-2 idiomorphs, which constitute the genetic control of O. sinensis reproduction, was analyzed in 237 strains and 5 genomes of H. sinensis. The mating-type genes were differentially transcribed in H. sinensis Strain L0106, and the MAT1-2-1 transcript in H. sinensis Strain 1229 was alternatively spliced, with an unspliced intron I containing stop codons; these findings constitute O. sinensis reproduction controls at the transcriptional and coupled transcriptional-translational levels. The occurrence of the mating-type genes and a-/α-pheromone receptor genes differed among the 5 H. sinensis genomes. The differential occurrence and transcription of these genes are inconsistent with self-fertilization under (pseudo)homothallism but suggest self-sterility of H. sinensis, which therefore needs a mating partner to achieve sexual outcrossing under heterothallism or hybridization. Consistent occurrence and transcription of the mating-type genes of both the MAT1-1 and MAT1-2 idiomorphs have been reported in natural and cultivated Cordyceps sinensis, but these findings are insufficient to determine the reproduction mode(s) because of the co-occurrence of >90 fungal species and multiple genomically independent genotypes of O. sinensis. The mutant MAT1-1-1 and α-pheromone receptor transcripts in natural C. sinensis result in the N-terminus or middle-truncated proteins, with changes in hydrophobicity and secondary structures of the proteins, suggesting heterogeneous fungal source(s) of the proteins and hybridization reproduction.

Coronavirus Disease-19 (COVID-19) is caused by the infection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The virus enters host cells through receptor-mediated endocytosis of angiotensin-converting enzyme-2 (ACE2), leading to systemic inflammation, also known as a “cytokine storm,” and neuroinflammation. COVID-19’s upstream regulator, interferon-gamma (IFNG), is downregulated upon the infection of SARS-CoV-2, which leads to the downregulation of ACE2. The neuroinflammation signaling pathway (NISP) can lead to neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by the formation of Lewy Bodies made of α-Synuclein protein encoded by synuclein alpha (SNCA). This study presents the pathways involved in the downregulation of ACE2 following SARS-CoV-2 infection and its effect on PD progression. Through QIAGEN’s Ingenuity Pathway Analysis, the study identifies the NISP as a top-five canonical pathway/signaling pathway and SNCA as a top-five upstream regulator. Core Analysis was also conducted on the associated molecules between COVID-19 and SNCA to construct a network connectivity map. The Molecule Activity Predictor tool was used to simulate the infection of SARS-CoV-2 by downregulating IFNG, which leads to the predicted activation of SNCA, and subsequently PD, through a dataset of intermediary molecules. Downstream Effect analysis was further used to quantify the downregulation of ACE2 on SNCA activation.

Extensive genome structure variation, such as copy number variations (CNVs) and presence/absence variations, is the basis for the remarkable maize genetic diversity; however, the effect of CNVs on maize herbivory defense remains largely underexplored. Here, we report that the naturally occurring duplication of the maize 9-lipoxygenase gene, ZmLOX5, leads to increased resistance of maize to herbivory by fall armyworms (FAW). Previously, we showed that ZmLOX5-derived oxylipins are required for defense against chewing insect herbivores and identified several inbred lines, including Yu796, that contained duplicated CNVs of ZmLOX5, referred to as Yu796-2×LOX5. To test whether introgression of the Yu796-2×LOX5 locus into the herbivore-susceptible B73 background that contains a single ZmLOX5 gene is a feasible approach to increase resistance, we generated a series of near-isogenic lines that contained either 2, 1 or 0 copies of the Yu796-2×LOX5 locus in the B73 background by six back crosses (BC6). Droplet digital PCR (ddPCR) confirmed the successful introgression of the Yu796-2×LOX5 locus in B73. The resulting B73-2×LOX5 inbred line displayed increased resistance against FAW associated with increased expression of ZmLOX5, increased wound-induced production of its primary oxylipin product, the α-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA), and the downstream defense hormones regulated by this molecule, 12-oxo-phytodienoic acid (12-OPDA) and abscisic acid (ABA). Surprisingly, wound-induced JA-Ile production was not increased in B73-2×LOX5, resulting from the increased JA catabolism. Furthermore, B73-2×LOX5 displayed enhanced drought tolerance likely due to increased ABA and 12-OPDA content. Taken together, this study revealed that the duplicated CNV of ZmLOX5 quantitively contributes to maize antiherbivore defense and presents proof-of-concept evidence that the introgression of naturally occurring duplicated CNVs of a defensive gene into the productive, but susceptible, crop varieties is a feasible breeding approach for enhancing plant resistance to herbivory and tolerance to abiotic stress.

Since the spread of the deadly virus SARS-CoV2 in late 2019, researchers have restlessly been seeking for unraveling how the virus factually enters the host cells. Some proteins on each side of the interaction between the virus and the host cells are involved as the major contributors to this process: 1- the nano-machine Spike protein on behalf of the virus, 2- angiotensin converting enzyme II, the mono-carboxypeptidase and the key component of renin angiotensin system on behalf of the host cell, 3- some host proteases and proteins exploited by SARS-CoV2, In this review, the complex process of SARS-CoV2 entrance into the host cells with the contribution of the involved host proteins as well as the sequential conformational changes in the Spike protein tending to increase the probability of complexification of the latter with angiotensin converting enzyme II, the receptor of the virus on the host cells, are discussed. Besides, the release of the catalytic ectodomain of angiotensin converting enzyme II as its soluble form in the extracellular space and its positive or negative impact on the infectivity of the virus are considered.