Protein Activity Research Articles

Evaluating the Potential of Green Light Exposure on Nociception-A Mini Review.

The capacity of animals to react to unpleasant stimuli that might endanger their integrity is known as nociception. Pharmacological treatments do not show satisfactory results in response to nociception. In the recent era, light therapy emerged as a potential non-pharmacological approach for treating various diseases, including seasonal affective disorders, migraine, pain, and others. Evaluating the potential of green light exposure on nociception involves studying its effects on different types of pain and pain-related conditions and determining the optimal exposure methods. This review provides the beneficial effects of green light on the reduction in the frequency of pain. The green light exposure on nociception changes the activity of pain-related genes and proteins in cells. This review could provide insights into the underlying mechanisms by which green light modulates pain. Overall, evaluating the potential of green light exposure on nociception requires a multidisciplinary approach and should consider the safety, efficacy, optimal dose, and duration of green light exposure and the type of pain. However, few studies have been reported so far; therefore, light therapy for treating migraines require more studies on animal models to provide precise results of light effects on nociception.

Role of TBK1 Inhibition in Targeted Therapy of Cancer.

TANK-binding kinase 1 (TBK1) is a serine/threonine protein that plays a crucial role in various biological processes like immunity, autophagy, cell survival, and proliferation. The level and kinase activity of the TBK1 protein is regulated through post-translational modifications (PTMs). TBK1 mainly mediates the activation of IRF3/7 and NF-κB signaling pathways while also participating in the regulation of cellular activities such as autophagy, mitochondrial metabolism, and cell proliferation. TBK1 regulates immune, metabolic, inflammatory, and tumor occurrence and development within the body through these cellular activities. TBK1 kinase has emerged as a promising therapeutic target for tumor immunity. However, its molecular mechanism of action remains largely unknown. The identification of selective TBK1 small molecule inhibitors can serve as valuable tools for investigating the biological function of TBK1 protein and also as potential drug candidates for tumor immunotherapy. The current research progress indicates that some TBK1 inhibitors (compounds 15,16 and 21) exhibit certain antitumor effects in vitro culture systems. Here, we summarize the mechanism of action of TBK1 in tumors in recent years and the progress of small molecule inhibitors of TBK1.

Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

In-silico approach to characterize the structure and function of a hypothetical protein of Monkeypox virus exploring Chordopox-A20R domain-containing protein activity.

Background: Monkeypox has emerged as a noteworthy worldwide issue due to its daily escalating case count. This illness presents diverse symptoms,including skin manifestations, which have the potential to spread through contact. The transmission of this infectious agent is intricate and readily transfers between individuals.Methods: The hypothetical protein MPXV-SI-2022V502225_00135 strain of monkeypox underwent structural and functional analysis using NCBI-CD Search, Pfam, and InterProScan. Quality assessment utilized PROCHECK, QMEAN, Verify3D, and ERRAT, followed by protein-ligand docking, visualization, and a 100-nanosecond simulation on Schrodinger Maestro.Results: Different physicochemical properties were estimated, indicating a stable molecular weight (49147.14) and theoretical pI (5.62) with functional annotation tools predicting the target protein to contain the domain of Chordopox_A20R domain. In secondary structure analysis, the helix coil was found to be predominant. The three-dimensional (3D) structure of the protein was obtained using a template protein (PDB ID:6zyc.1), which became more stable after YASARA energy minimization and was validated by quality assessment tools like PROCHECK, QMEAN, Verify3D, and ERRAT. Protein-ligand docking was conducted using PyRx 9.0 software to examine the binding and interactions between a ligand and a hypothetical protein, focusing on various amino acids. The model structure, active site, and binding site were visualized using the CASTp server, FTsite, and PyMOL. A 100 nanosecond simulation was performed with ligand CID_16124688 to evaluate the efficiency of this protein.Conclusion: The analysis revealed significant binding interactions and enhanced stability, aiding in drug or vaccine design for effective antiviral treatment and patient management.

Advance in Iron Metabolism, Oxidative Stress and Cellular Dysfunction in Experimental and Human Kidney Diseases

Kidney diseases pose a significant global health issue, frequently resulting in the gradual decline of renal function and eventually leading to end-stage renal failure. Abnormal iron metabolism and oxidative stress-mediated cellular dysfunction facilitates the advancement of kidney diseases. Iron homeostasis is strictly regulated in the body, and disturbance in this regulatory system results in abnormal iron accumulation or deficiency, both of which are associated with the pathogenesis of kidney diseases. Iron overload promotes the production of reactive oxygen species (ROS) through the Fenton reaction, resulting in oxidative damage to cellular molecules and impaired cellular function. Increased oxidative stress can also influence iron metabolism through upregulation of iron regulatory proteins and altering the expression and activity of key iron transport and storage proteins. This creates a harmful cycle in which abnormal iron metabolism and oxidative stress perpetuate each other, ultimately contributing to the advancement of kidney diseases. The crosstalk of iron metabolism and oxidative stress involves multiple signaling pathways, such as hypoxia-inducible factor (HIF) and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways. This review delves into the functions and mechanisms of iron metabolism and oxidative stress, along with the intricate relationship between these two factors in the context of kidney diseases. Understanding the underlying mechanisms should help to identify potential therapeutic targets and develop novel and effective therapeutic strategies to combat the burden of kidney diseases.

Metabolic and productive parameters of lactating dairy cows under heat stress conditions supplemented with plant polyphenol extract.

The impact of heat stress on dairy cattle leads to significant economic losses and a negative impact on the welfare of the animals. The objective of this research was to evaluate the effect of the nutritional additive (Thermoplus®) in dairy cows under postpartum heat stress conditions, and its effects on the metabolic profile, production and quality of milk. Eighteen lactating Holstein cows (8 multiparous and ten primiparous), in a free-stall system, with a mean body condition score (BCS) of 3.14 ± 0.05, live weight of 624.55 ± 18, 61kg, with initial mean days in milk (DIM) of 90 ± 10.11, were selected. The animals were grouped into a control (CG, n = 9) and a treatment (TG, n = 9). Both groups underwent 14days of diet adaptation, the TG received the basal diet supplemented with 50g of the additive, once a day, individually, while the control group received only the total diet. Data collection of metabolic and productive parameters were evaluated on days -14 (before adaptation), 1 (after the diet adaptation period), 16, 30, and 44. Milk, blood, and body condition score (BCS) were collected once a day, and heart rate, respiratory rate, and rectal temperature were collected twice a day. Serum concentrations of albumin, calcium, magnesium, glucose, gamma-glutamyl transferase (GGT), beta-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFAs), and paraoxonase-1 (PON-1) were evaluated. In the milk, the percentage of fat, protein, lactose, and total solids were determined in each sampling. Milk yield was measured daily. Humidity and ambient temperature values were collected on the days of the collection every 30min, from 5:30 am to 5:00pm, to calculate the temperature-humidity index (THI). Statistical analyzes were performed using the SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). The THI ranged from 62.22 to 79.47. Our findings showed that when the THI was greater than 72, the animals in the TG were able to maintain milk yield (Odds ratio (OD) = -0.0577,), and the animals in the CG had a greater chance of reducing it (OD = -0.2301). Multiparous cows in the TG had higher milk yield than CG (32.57 ± 0.34 vs 30.50 ± 0.36kg per day; P = 0.0078) and lower SCC (34.110 ± 6,940 vs 665.50 ± 214.41 cells per ml; P = 0.03), with the same percentages of total solids (P > 0.05). In multiparous metabolic markers, TG when compared CG had higher albumin concentrations (2.50 ± 0.07 vs 2.12 ± 0.07g/dl; < 0.001), equal PON-1 (P > 0.05), and higher BHBA levels (0.49 ± 0.03 vs 0.39 ± 0.04mmol/l). Primiparous from the CG had higher concentrations of NEFA (0.18 ± 0.02mmol/l) than multiparous from the same group (0.09 ± 0.02mmol/l) P = 0.0265. The use of the plant polyphenol extract in postpartum Holstein cows challenged by heat stress had beneficial effects on the production and health of the mammary gland in multiparous cows without decreasing milk solids. The non-reduction of the activities of the acute phase proteins indicates an immunomodulatory and inflammatory-reducing effect of the product used.

Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Conserved quality control mechanisms of mitochondrial protein import.

Mitochondria carry out essential functions for the cell, including energy production, various biosynthesis pathways, formation of co-factors and cellular signalling in apoptosis and inflammation. The functionality of mitochondria requires the import of about 900-1300 proteins from the cytosol in baker's yeast Saccharomyces cerevisiae and human cells, respectively. The vast majority of these proteins pass the outer membrane in a largely unfolded state through the translocase of the outer mitochondrial membrane (TOM) complex. Subsequently, specific protein translocases sort the precursor proteins into the outer and inner membranes, the intermembrane space and matrix. Premature folding of mitochondrial precursor proteins, defects in the mitochondrial protein translocases or a reduction of the membrane potential across the inner mitochondrial membrane can cause stalling of precursors at the protein import apparatus. Consequently, the translocon is clogged and non-imported precursor proteins accumulate in the cell, which in turn leads to proteotoxic stress and eventually cell death. To prevent such stress situations, quality control mechanisms remove non-imported precursor proteins from the TOM channel. The highly conserved ubiquitin-proteasome system of the cytosol plays a critical role in this process. Thus, the surveillance of protein import via the TOM complex involves the coordinated activity of mitochondria-localized and cytosolic proteins to prevent proteotoxic stress in the cell.

Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems.

The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.

Spasmolytic, Antimicrobial, and Antioxidant Activities of Spray-Dried Extracts of Gentiana asclepiadea L. with In Silico Pharmacokinetic Analysis

This study aimed to evaluate the spasmolytic activity of an underground parts extract of Gentiana asclepiadea L. (Gentianaceae), assess its antioxidant and antimicrobial activities, and explore the impact of extract encapsulation on the aforementioned bioactivities. An extract encapsulated by spray drying with whey protein, pure extract, and pure whey protein were comparatively tested. The main compounds identified via HPLC-DAD analysis underwent in silico ADME assessment. The spasmolytic effect was tested on a model of spontaneous rat ileum contractions, and the mechanism of action was further evaluated on acetylcholine-, KCl-, CaCl2-, BaCl2-, histamine-, N(ω)-nitro-L-arginine methyl ester-, and glibenclamide-modified contractions. The most abundant compounds were secoiridoids (dominantly gentiopicroside), followed by C-glycosylated flavonoids and xanthones. Both pure and encapsulated extracts achieved significant spasmolytic effects, despite the spasmogenic activity of pure whey protein. The extract may exert its spasmolytic effect through multiple pathways, predominantly by antagonizing the Ca2+ channel and opening the K+ channel, while the nitric oxide pathway appears not to be involved. The antimicrobial and antioxidant activities of the pure extract were moderate. The extract stabilized by encapsulation retained all of the tested bioactivities of the unencapsulated extract. The obtained results suggest that G. asclepiadea has potential for use in the treatment of some gastrointestinal complaints and that the encapsulated extract could be a valuable functional ingredient in pharmaceutical and food products.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Computational analysis of propeptide-containing proteins and prediction of their post-cleavage conformation changes.

A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and activation and are present in about 2.3% of reviewed proteins in the UniProt database. They are often found in secreted or membrane-bound proteins including proteolytic enzymes, hormones, and toxins. We identified a variety of globular and nonglobular Pfam domains in protein sequences designated as propeptides, some of which form intramolecular interactions with other domains in the mature proteins. Propeptide-containing enzymes mostly function as proteases, as they are depleted in other enzyme classes such as hydrolases acting on DNA and RNA, isomerases, and lyases. We applied AlphaFold to generate structural models for over 7000 proteins with propeptides having no less than 20 residues. Analysis of residue contacts in these models revealed conformational changes for over 300 proteins before and after the cleavage of the propeptide. Examples of conformation change occur in several classes of proteolytic enzymes in the families of subtilisins, trypsins, aspartyl proteases, and thermolysin-like metalloproteases. In most of the observed cases, cleavage of the propeptide releases the constraints imposed by the covalent bond between the propeptide and the mature protein, and cleavage enables stronger interactions between the propeptide and the mature protein. These findings suggest that post-cleavage propeptides could play critical roles in regulating the activity of mature proteins.

Ciliary tip actin dynamics regulate photoreceptor outer segment integrity

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line

BackgroundThe burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa.MethodsThe proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay.ResultsThe crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity.ConclusionThis study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

Machine learning-assisted SERS approach enables the biochemical discrimination in Bcl-2 and Mcl-1 expressing yeast cells treated with ketoconazole and fluconazole antifungals

Antifungal medications are important due to their potential application in cancer treatment either on their own or with traditional treatments. The mechanisms that prevent the effects of these medications and restrict their usage in cancer treatment are not completely understood. The evaluation and discrimination of the possible protective effects of the anti-apoptotic members of the Bcl-2 family of proteins, critical regulators of mitochondrial apoptosis, against antifungal drug-induced cell death has still scientific uncertainties that must be considered. Novel, simple, and reliable strategies are highly demanded to identify the biochemical signature of this phenomenon. However, the complex nature of cells poses challenges for the analysis of cellular biochemical changes or classification. In this study, for the first time, we investigated the probable protective activities of Bcl-2 and Mcl-1 proteins against cell damage induced by ketoconazole (KET) and fluconazole (FLU) antifungal drugs in a yeast model through surface-enhanced Raman spectroscopy (SERS) approach. The proposed SERS platform created robust Raman spectra with a high signal-to-noise ratio. The analysis of SERS spectral data via advanced unsupervised and supervised machine learning methods enabled unquestionable differentiation (100 %) in samples and biomolecular identification. Various SERS bands related to lipids and proteins observed in the analyses suggest that the expression of these anti-apoptotic proteins reduces oxidative biomolecule damage induced by the antifungals. Also, cell viability assay, Annexin V-FITC/PI double staining, and total oxidant and antioxidant status analyses were performed to support Raman measurements. We strongly believe that the proposed approach paves the way for the evaluation of various biochemical structures/changes in various cells.

Clinical and functional characterization of a novel KCNJ11 (c.101G > A, p.R34H) mutation associated with maturity-onset diabetes mellitus of the young type 13.

This study aimed to describe the clinical features, diagnostic and therapeutic course of a patient with MODY13 caused by KCNJ11 (c.101G > A, p.R34H) and how it contributes to the pathogenesis of MODY13, and to explore new therapeutic targets. Whole-exome sequencing was used to screen prediagnosed individuals and family members with clinically suspected KCNJ11 mutations. Real-time fluorescence quantitative PCR, western blotting, thallium flux of potassium channels, glucose-stimulated insulin secretion (GSIS), and immunofluorescence assays were used to analyze the regulation of insulin secretion by the KCNJ11 mutant in MIN6 cells. Daily blood glucose levels were continuously monitored for 14 days in the proband using the ambulatory blood glucose meter (SIBIONICS). Mutation screening of the entire exon of the gene identified a heterozygous KCNJ11 (c.101G > A, p.R34H) mutation in the proband and his mother. Cell-based GSIS assays after transfection of MIN6 using wild-type and mutant plasmids revealed that this mutation impaired insulin secretory function. Furthermore, we found that this impaired secretory function is associated with reduced functional activity of the mutant KCNJ11 protein and reduced expression of the insulin secretion-associated exocytosis proteins STXBP1 and SNAP25. For the first time, we revealed the pathogenic mechanism of KCNJ11 (c.101G > A, p.R34H) associated with MODY13. This mutant can cause alterations in KATP channel activity, reduce sensitivity to glucose stimulation, and impair pancreatic β-cell secretory function by downregulating insulin secretion-associated exocytosis proteins. Therefore, oral sulfonylurea drugs can lower blood glucose levels through pro-insulinotropic effects and are more favorable for patients with this mutation.

Identification of disease-specific genes related to immune infiltration in nonalcoholic steatohepatitis using machine learning algorithms.

To identify disease signature genes associated with immune infiltration in nonalcoholic steatohepatitis (NASH), we downloaded 2 publicly available gene expression profiles, GSE164760 and GSE37031, from the gene expression omnibus database. These profiles represent human NASH and control samples and were used for differential genes (DEGs) expression screening. Two machine learning methods, the Least Absolute Shrinkage and Selection Operator regression model and Support Vector Machine Recursive Feature Elimination, were used to identify candidate disease signature genes. The CIBERSORT deconvolution algorithm was employed to analyze the infiltration of 22 immune cell types in NASH. Additionally, we constructed a NASH cell model using HepG2 cells treated with oleic acid and free fatty acids. The construction of the cell model was verified using oil red O staining, and Western blotting was used to detect the protein expression of the disease signature genes in both control and model groups. As a result, a total of 262 DEGs were identified. These DEGs were primarily associated with metal ion transmembrane transporter activity, sodium ion transmembrane transporter protein activity, calcium ion, and neuroactive ligand-receptor interactions. FOS, IGFBP2, dual-specificity phosphatase 1 (DUSP1), and IKZF3 were identified as disease signature genes of NASH by the least absolute shrinkage and selection operator and Support Vector Machine Recursive Feature Elimination algorithms for DEGs analysis. The receiver operating characteristic curves showed that FOS, IGFBP2, DUSP1, and IKZF3 had good diagnostic value (area under receiver operating characteristic curve > 0.8). These findings were validated in the GSE89632 dataset and through cellular assays. Immunocyte infiltration analysis revealed that NASH was associated with CD8 T cells, CD4 T cells, follicular helper T cells, resting NK cells, eosinophils, regulatory T cells, and γδ T cells. The FOS, IGFBP2, DUSP1, and IKZF3 genes were specifically associated with follicular helper T cells. Lipid droplet aggregation significantly increased in HepG2 cells treated with oleic acid and free fatty acids, indicating successful construction of the cell model. In this model, the expression of FOS, IGFBP2, and DUSP1 was significantly decreased, while that of IKZF3 was significantly elevated (P < .01, P < .001) compared with the control group. Therefore, FOS, IGFBP2, DUSP1, and IKZF3 can be considered as disease signature genes associated with immune infiltration in NASH.

Curcumin Nanogel Preparations: A Promising Alternative for Psoriasis Treatment.

Curcumin is a naturally occurring polyphenolic compound extracted from the rhizomes of Curcuma longa, commonly known as turmeric. It has been used for centuries in traditional medicine and is gaining increasing attention in modern medicine owing to its potential therapeutic benefits. Psoriasis is a chronic inflammatory disease characterized by red scaly patches on the skin. Curcumin has been found to be effective in treating psoriasis by inhibiting the activity of various enzymes and proteins involved in the inflammation and proliferation of psoriatic skin cells. Nanogel preparation of curcumin has been found to be a promising approach for the delivery of compounds to treat psoriasis. Nanogels are composed of biocompatible and biodegradable crosslinked hydrogels. The nanogel formulation of curcumin increases its solubility, stability, and bioavailability, indicating that a lower dose is needed to achieve the same therapeutic effect. This review article suggests that the nanogel preparation of curcumin can be a better alternative for psoriasis treatment as it increases the bioavailability and stability of curcumin and also reduces the required dosage. This study suggests that curcumin nanogel preparations are promising alternatives to traditional psoriasis treatments and could potentially be used as a more effective and safe treatment option. This article highlights the need for further research to fully understand the potential of curcumin nanogel preparations for psoriasis treatment in humans.