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Background: Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder in children and adolescents characterized by persistent patterns of hyperactivity, impulsivity, and inattentiveness. ADHD persists for many into adulthood. While irritability is not a diagnostic symptom of ADHD, temper outbursts and irritable moods are common in individuals with ADHD. However, research on the association between irritability and ADHD symptoms in adolescents and young adults remains limited., Method: Prior research has used linear regression models to examine longitudinal relations between ADHD and irritability symptoms. This method may be impacted by the potential presence of highly colinear variables. We utilized a hierarchical clustering technique to mitigate these collinearity issues and implemented a non-parametric machine learning (ML) model to predict the significance of symptom relations over time. Our data included adolescents (N=148, 54% ADHD) and young adults (N=124, 42% ADHD) diagnosed with ADHD and neurotypical (NT) individuals, evaluated in a longitudinal study., Results: Results from the linear regression analysis indicate a significant association between irritability at time-point 1 (T1) and hyperactive-impulsive symptoms at time-point 2 (T2) in adolescent females (β=0.26, p-value < 0.001), and inattentiveness at T1 with irritability at T2 in young adult females (β=0.49, p-value < 0.05). Using a non-parametric-based approach, employing the Random Forest (RF) method, we found that among both adolescents and young adults, irritability in adolescent females significantly contributes to predicting impulsive symptoms in subsequent years, achieving a performance rate of 86%., Conclusion: Our results corroborate and extend prior findings, allowing for an in-depth examination of longitudinal relations between irritability and ADHD symptoms, namely hyperactivity, impulsivity, and inattentiveness, and the unique association between irritability and ADHD symptoms in females., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Komijani, Ghosal, Singh, Schweitzer and Mukherjee.)

Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in E. coli Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05-1 mM), cell density (OD 600 : 0.45-0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0-10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD 600: 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is an aggressive blood cancer with frequently poor clinical outcomes. This heterogeneous malignancy encompasses genetically, molecularly, and even clinically different subgroups. This makes it difficult to develop therapeutic agents that are effective for all subtypes of the disease. Therefore, a selective, universal, and adaptable delivery platform capable of carrying various types of anti-neoplastic agents is an unmet requirement in this area. Two multifunctional fusion proteins were designed for the delivery of biotinylated cargoes to human myeloid leukemia cells by fusing an anti-IL-1RAP single-chain antibody with streptavidin (tetramer or monomer), a cell-penetrating peptide (CPP), and an endosomolytic peptide in a single biomacromolecule. The designed fusions were analyzed primarily in silico, and the biofunctionality of the selected fusion was fully characterized via several binding assays, hemolysis assay, confocal microscopy and cell cytotoxicity assay after production via the Escherichia coli (E. coli) system. The refolded protein exhibited desirable binding activity to leukemic cells, pure antigen and biotinylated BSA. Further analyses revealed efficient cellular uptake, endosomolytic activity, and nuclear penetration without any detectable cytotoxicity toward normal epithelial cells. The described platform seems to have great potential for targeted delivery of different therapeutics to malignant myeloid cells., (© 2024. The Author(s).)

We report the synthesis of functionalized cycloheptanes by thermal electrocyclization of heptatrienyl anions under mild conditions. In addition, we disclose the first examples of this electrocyclization manifold conducted under catalytic conditions. Previously, electrocyclization of heptatrienyl systems required formation of anions with a strong base, resulting in limited functional group compatibility. We demonstrate that polarization of heptatrienyl anions using strategically positioned electron-withdrawing groups lowers the energy landscape of the reaction by stabilizing both the acyclic heptatrienyl anion and cycloheptadienyl product. Divergent reactivity is observed between aliphatic and aromatic substrates, with the latter requiring only catalytic amounts of base for complete conversion. This can be rationalized by the relative stability of the acyclic and cyclic anions and their ability to participate in a chain reaction process., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In this work, a magnetic nanobiocomposite scaffold based on carboxymethylcellulose (CMC) hydrogel, silk fibroin (SF), and magnetite nanoparticles was fabricated. The structural properties of this new magnetic nanobiocomposite were characterized by various analyses such as FT-IR, XRD, EDX, FE-SEM, TGA and VSM. According to the particle size histogram, most of the particles were between 55 and 77 nm and the value of saturation magnetization of this nanobiocomposite was reported 41.65 emu.g - 1 . Hemolysis and MTT tests showed that the designed magnetic nanobiocomposite was compatible with the blood. In addition, the viability percentage of HEK293T normal cells did not change significantly, and the proliferation rate of BT549 cancer cells decreased in its vicinity. EC50 values for HEK293T normal cells after 48 h and 72 h were 3958 and 2566, respectively. Also, these values for BT549 cancer cells after 48 h and 72 h were 0.4545 and 0.9967, respectively. The efficiency of fabricated magnetic nanobiocomposite was appraised in a magnetic fluid hyperthermia manner. The specific absorption rate (SAR) of 69 W/g (for the 1 mg/mL sample at 200 kHz) was measured under the alternating magnetic field (AMF)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs)., Methods: The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins., Results: By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G 4 S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G 4 S molecule., Conclusions: Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein., Competing Interests: Conflict of Interests: There is no potential conflict of interests., (Copyright © 2023, Author(s).)

Background: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli)., Methods: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum., Results: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance 450nm ). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability., Conclusion: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In this study, the main focus was on designing and synthesizing a novel magnetic nanobiocomposite and its application in hyperthermia cancer treatment. Regarding this aim, sodium alginate (SA) hydrogel with CaCl 2 cross-linker formed and modified by silk fibroin (SF) natural polymer and halloysite nanotubes (HNTs), followed by in situ Fe 3 O 4 magnetic nanoparticles preparation. No important differences were detected in red blood cells (RBCs) hemolysis, confirming the high blood compatibility of the treated erythrocytes with this nanobiocomposite. Moreover, the synthesized SA hydrogel/SF/HNTs/Fe 3 O 4 nanobiocomposite does not demonstrate toxicity toward HEK293T normal cell line after 48 and 72 h. The anticancer property of SA hydrogel/SF/HNTs/Fe 3 O 4 nanobiocomposites against breast cancer cell lines was corroborated. The magnetic saturation of the mentioned magnetic nanobiocomposite was 15.96 emu g -1 . The specific absorption rate (SAR) was measured to be 22.3 W g -1 by applying an alternating magnetic field (AMF). This novel nanobiocomposite could perform efficiently in the magnetic fluid hyperthermia process, according to the obtained results., (© 2022. The Author(s).)

Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS., Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours)., Results: : Increased efficiency was achieved by EnBase® compared to Luria–Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 μg/mL., Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.