Full Length Research Articles

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K‑562 and colorectal HCT‑116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme‑dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM‑sensitive K‑562 chronic myeloid leukemia cells (K‑562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K‑562 and IM‑chemoresistant K‑562 chronic myeloid leukemia cells (K‑562R), as well as human colorectal carcinoma cells HCT‑116 (+/+p53) and (‑/‑p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2µM) and/or DCA (4mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde‑3‑phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor‑1a, heme oxygenase‑1, NF‑κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K‑562 and considerable (>45%) in HCT‑116 (+/+p53) cultures, but less in K‑562R and HCT‑116 (‑/‑p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow‑cytometry, in K‑562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

Molecular mechanism of bovine Gasdermin D-mediated pyroptosis

Pyroptosis is a form of programmed cell death characterized by cell swelling, pore formation in the plasma membrane, lysis, and releases of cytoplasmic contents. To date, the molecular mechanism of human and murine Gasdermin D-mediated pyroptosis have been fully investigated. However, studies focusing on molecular mechanism of bovine Gasdermin D (bGSDMD)-mediated pyroptosis and its function against pathogenic infection were unclear. In the present study, we demonstrate that bovine caspase-1 (bCaspase-1) cleaves bGSDMD at amino acid residue D277 to produce an N-terminal fragment (bGSDMD-p30) which leads to pyroptosis. The amino acid residues T238 and F239 are critical for bGSDMD-p30-mediated pyroptosis. The loop aa 278-299, L293 and A380 are the key sites for autoinhibitory structure of the full length of bGSDMD. In addition, bCaspase-3 also cleaves bGSDMD at residue Asp86 without inducing cell death. Therefore, our study provides the first detailed elucidation of the mechanism of bovine GSDMD-mediated pyroptosis. The results will establish a significant foundation for future research on the role of pyroptosis in bovine infectious diseases.

Unsteady solute transport in Casson fluid flow and its retention in an atherosclerotic wall

In the current study, a mathematical model of convective solute transport and its retention in an atherosclerotic wall is examined. The artery wall is supposed to be permeable and has cosine-shaped stenosis caused by various sorts of abnormal growth or plaque formation. The flowing blood is modeled as the suspension of all red blood cells in plasma, thought to be Casson fluid. The Immersed Boundary Method (IBM) and the Marker and Cell (MAC) method are used to numerically solve the unsteady governing equations of motion along with the appropriate initial and boundary conditions. The final results of the quantitative studies contain the corresponding flow-field and solute distribution profiles for the full arterial length. The essential components are evaluated, including the wall shear stress, wall concentration, and concentration contours. According to simulation data, the permeability of the wall directly affects the solute transport within the therapeutic region. The yield stress does, however, increase flow resistance and decrease solute uptake within the tissue. The projected outcomes are remarkably in line with previously published findings, which supports the applicability of the model under investigation.

Renal Expression and Localization of the Receptor for (Pro)renin and Its Ligands in Rodent Models of Diabetes, Metabolic Syndrome, and Age-Dependent Focal and Segmental Glomerulosclerosis.

The (pro)renin receptor ((P)RR), a versatile protein found in various organs, including the kidney, is implicated in cardiometabolic conditions like diabetes, hypertension, and dyslipidemia, potentially contributing to organ damage. Importantly, changes in (pro)renin/(P)RR system localization during renal injury, a critical information base, remain unexplored. This study investigates the expression and topographic localization of the full length (FL)-(P)RR, its ligands (renin and prorenin), and its target cyclooxygenase-2 and found that they are upregulated in three distinct animal models of renal injury. The protein expression of these targets, initially confined to specific tubular renal cell types in control animals, increases in renal injury models, extending to glomerular cells. (P)RR gene expression correlates with protein changes in a genetic model of focal and segmental glomerulosclerosis. However, in diabetic and high-fat-fed mice, (P)RR mRNA levels contradict FL-(P)RR immunoreactivity. Research on diabetic mice kidneys and human podocytes exposed to diabetic glucose levels suggests that this inconsistency may result from disrupted intracellular (P)RR processing, likely due to increased Munc18-1 interacting protein 3. It follows that changes in FL-(P)RR cellular content mechanisms are specific to renal disease etiology, emphasizing the need for consideration in future studies exploring this receptor's involvement in renal damage of different origins.

Olympic Weightlifting Training for Sprint Performance in Athletes: A Systematic Review with Meta-analysis.

This systematic review and meta-analysis aimed to determine whether Olympic weightlifting (OW) exercises would improve sprint performance when compared to a control intervention, (no training, standard sport-specific training, traditional resistance training, or plyometric training). Medline, Web of Science, SportDiscus, CINAHL, and Biological Science from inception to September 2022 was searched. Two authors independently selected the included studies, extracted data, and appraised the risk of bias. Certainty of the evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation methodology. The primary meta-analysis combined the results of the sprint performance over the full length of each sprint test. The secondary meta-analyses combined the results of the sprint performance at 5, 10, and 20 m distance to capture information about the acceleration phase of the sprint tests. Eight studies with 206 athletes (female n=10, age range: 18.9-24.2 years) were identified. Sprint performance did not differ significantly comparing OW to the control intervention, nor at the full length (standardized mean difference=-0.07, 95% CI=-0.47 to 0.34, p=0.75, I2=46%) or during the acceleration phase (p≥0.26) of the sprint test. OW training does not improve sprint performance to a greater extent than comparator interventions.

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BackgroundCampylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide.MethodsWe systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids.ResultsOverall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter.ConclusionsThe results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Synthesis of Self Permeable Antisense PMO Using C5-Guanidino-Functionalized Pyrimidines at the 5'-End Enables Sox2 Downregulation in Triple Negative Breast Cancer Cells.

Delivery of macromolecular drugs inside cells has been a huge challenge in the field of oligonucleotide therapeutics for the past few decades. Earliest natural inspirations included the arginine rich stretch of cell permeable HIV-TAT peptide, which led to the design of several molecular transporters with varying numbers of rigid or flexible guanidinium units with different tethering groups. These transporters have been shown to efficiently deliver phosphorodiamidate morpholino oligonucleotides, which have a neutral backbone and cannot form lipoplexes. In this report, PMO based delivery agents having 3 or 4 guanidinium groups at the C5 position of the nucleobases of cytosine and uracil have been explored, which can be assimilated within the desired stretch of the antisense oligonucleotide. Guanidinium units have been connected by varying the flexibility with either a saturated (propyl) or an unsaturated (propargyl) spacer, which showed different serum dependency along with varied cytoplasmic distribution. The effect of cholesterol conjugation in the delivery agent as well as at the 5'-end of full length PMO in cellular delivery has also been studied. Finally, the efficacy of the delivery has been studied by the PMO mediated downregulation of the stemness marker Sox2 in the triple-negative breast cancer cell line MDA-MB 231. These results have validated the use of this class of delivery agents, which permit at a stretch PMO synthesis where the modified bases can also participate in Watson-Crick-Franklin base pairing for enhanced mRNA binding and protein downregulation and could solve the delivery problem of PMO.

Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BackgroundRheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear.MethodsBased on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed.ResultsComparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity.ConclusionRpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

(128) Characterizing Antibiotic Susceptibility of the Penile Prosthesis Microbiome

Abstract Introduction Inflatable penile prostheses (IPP) are the gold standard treatment for erectile dysfunction refractory to conservative therapies. However, infection remains a significant concern and the presence of microbial biofilms on both infected and non-infected devices has recently been demonstrated. Various antimicrobials are used for prophylaxis intraoperatively and postoperatively. It remains unknown how these antimicrobials affect the growth of bacteria and their associated biofilms. We sought to assess how commonly used antimicrobials influenced growth of bacterial isolates previously isolated from both non-infected and infected devices, obtained during revisional surgery. Objective The goal of this study was to evaluate growth of bacterial isolates previously obtained from non-infected and infected IPPs. Isolates were exposed to single or multi-antibiotic combinations to assess degree of inhibition of bacterial growth. Methods In this IRB-approved study, 35 bacterial isolates had been previously obtained from patients who had been consented for IPP revision or replacement secondary to mechanical failure, pain, or device infection. Bacteria were plated on brain-heart-infusion (BHI) agar plates and allowed to incubate for 48 hours. Standard-sized antibiotic discs were introduced into the grown bacterial colonies consisting of the following single or multi-agent regimens: bacitracin, cefaclor, cephazolin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tobramycin, vancomycin, piperacillin/tazobactam, gentamicin + piperacillin/tazobactam, gentamicin + cephazolin, and gentamicin + vancomycin. Zones of inhibition (ZOI) were measured after placement of the antibiotic discs in the bacterial colonies. Statistics were analyzed with false discovery rate corrected, one-sample t-tests. Quantification of antibiotic resistance was defined as follows: Resistant: ZOI not significantly different from 0 in one sample t-test Susceptible: ZOI significantly greater than 0, but mean is <10mm Highly susceptible: ZOI significantly greater than 0, but mean is 10-20mm Exceptionally susceptible: ZOI significantly greater than 0, but mean is >20mm Taxonomic classification of bacterial isolates was provided by extracting DNA from pure cultures and sequencing the full length 16S rRNA gene using Sanger sequencing, after amplification with two different primer sets to provide overlapping sequence regions. Results Of the antibiotics tested, TMP-SMX was found to have the highest percentage of resistant isolates (54.3%) of the 35 tested, with bacitracin having the second most (40%). Both tobramycin and TMP-SMX had 0.0% of isolates be exceptionally susceptible. Combination piperacillin/tazobactam was found to have the highest percentage of exceptionally susceptible isolates (11.4%). A large majority of isolates were highly susceptible to gentamicin + piperacillin/tazobactam (91.4%), piperacillin/tazobactam (74.3%), gentamicin + cefazolin (74.3%), and levofloxacin (88.6%), while greater than half of isolates were highly susceptible to cefazolin (62.9%) and cefaclor (54.3%)." Conclusions Our results demonstrate that cefaclor or bacitracin prevented growth of bacterial isolates obtained from IPPs explanted for mechanical failure, pain or infection. Further investigation is needed to characterize how intraoperative and postoperative antibiotic influences a symbiotic or dysbiotic community of microbes on the surface of penile prostheses. By better understanding the interactions between microbes, antibiotics and host, novel therapeutics might promote a non-infected, symbiotic microenvironment. Disclosure Yes, this is sponsored by industry/sponsor: Coloplast corporation. Clarification: Industry funding only - investigator initiated and executed study. Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast, boston scientific.

Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-β peptide (Aβ) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

The Effect of Oral Motor Stimulation on the Transition to Full Oral Feeding, Breastfeeding, and Length of Hospital Stay in Preterm Infants.

Objective: To determine the effect of oral motor stimulation (OMS) applied to preterm infants on their sucking and swallowing abilities to establish a successful and safe oral feeding experience. Methods: A pre-post intervention study was conducted between December 2019 and December 2020, which included preterm infants born at <35 weeks of gestational age and admitted to the neonatal intensive care unit. Patients with major congenital abnormalities (including cardiac, facial, and jaw deformities), severe NEC, stage 3-4 IVH were excluded from the study. Patients who received OMS by a speech and language therapist between June 2020 and December 2020 were assigned to Group 1, while patients who received no intervention between December 2019 and May 2020 were assigned to Group 2. The time to achieve full oral feeding (FOF), acquisition of breastfeeding rates at discharge, and the length of hospital stay (LOS) were compared between the groups. Results: A total of 62 patients were included in the study (31 in Group 1 and 31 in Group 2). There were no significant differences in birth weight and demographic data between the groups. The mean time to achieve FOF was found to be significantly shorter in Group 1 (31 ± 23.6 and 46.7 ± 22.3 days, respectively, p = 0.013). The mean LOS was also found to be shortened with a mean duration of 10 days in Group 1, without statistical significance (56.4 ± 35.3 days versus 66.0 ± 42.9 days, respectively, p = 0.34). Acquisition of breastfeeding rates was significantly higher in the intervention group (p < 0.05) Conclusions: OMS accelerates the transition to FOF in preterm infants and increases the rates of acquiring breastfeeding skills at discharge.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Structural insights into the unique recognition module between α-synuclein peptide and nanobody.

Nanobodies are single-domain fragments of antibodies with comparable specificity and affinity to antibodies. They are emerging as versatile tools in biology due to their relatively small size. Here, we report the crystal structure of a specific nanobody Nbα-syn01, bound to a 14 amino acid long peptide of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. The complex structure exhibits a unique binding pattern where the αSyn peptide replaces the N-terminal region of nanobody. Recognition is mediated principally by extended main chain interaction of the αSyn peptide and specificity of the interaction lies in the central 48-52 region of αSyn peptide. Structure-guided truncation of Nbα-syn01 shows tighter binding to αSyn peptide and improved inhibition of α-synuclein aggregation. The structure of the truncated complex was subsequently determined and was indistinguishable to full length complex as the full-length form had no visible electron density for the N-terminal end. These findings reveal the molecular basis for a previously unobserved binding mode for nanobody recognition of α-synuclein, providing an explanation for the enhanced binding, and potential for an alternate framework for structure-based protein engineering of nanobodies to develop better diagnostic and therapeutic tools.

Experimental and numerical investigation on the crashworthiness performance of double hat‐section Al‐CFRP beam subjected to quasi‐static bending test

AbstractNowadays, hybrid structures, which combine low‐density composites with low‐cost thin‐walled metals, have shown great effect in enhancing the performance of automotive body structures, especially the energy absorber components. This study focuses on the experimental and numerical investigation of the bending energy absorption behavior of CFRP/aluminum hybrid double hat‐section beams used in the side part of the car body. In the experimental section, two types of double hat‐section beams were fabricated: aluminum and Al/CFRP. These beams were then subjected to quasi‐static three‐point bending tests. The results demonstrated that the hybrid beam exhibited superior energy absorption capabilities compared to the single beam. Subsequently, a finite element model was developed using LS‐Dyna software and was validated by comparing the experimental and numerical load–displacement results. In‐depth numerical analyses were conducted to investigate the influence of various design parameters, including CFRP‐reinforcement configuration, numbers of CFRP layers, CFRP ply‐angle, CFRP reinforced length, and aluminum/CFRP mass, on crashworthiness indicators. The numerical findings indicate that the hybrid beam with , ply‐angles exhibited better crash force efficiency (CFE) and specific energy absorption (SEA) compared to other ply‐angles, respectively. Furthermore, increasing the number of CFRP layers within the total beam's mass improved its energy absorption capacity. It was also revealed that the CFRP length on the upper and lower hats could be considered as 42.5% of the total aluminum beam length, as opposed to the full CFRP length, providing enhanced energy absorption capabilities.Highlights Bending behavior of the Al and Al/CFRP double hat section beams. Effects of variable thickness and identical mass on the double hat section beam. Effects of variable ply angle and number of CFRP layers on the hybrid beams. Discrete effect of CFRP reinforcement configurations and inner CFRP lengths.

AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis

Many types of tumors feature aerobic glycolysis for meeting their increased energetic and biosynthetic demands. However, it remains still unclear how this glycolytic phenomenon is achieved and coordinated with other metabolic pathways in tumor cells in response to growth stimuli. Here we report that activation of AKT1 induces a metabolic switch to glycolysis from the mitochondrial metabolism via phosphorylation of cytoplasmic malic enzyme 2 (ME2), named ME2fl (fl means full length), favoring an enhanced glycolytic phenotype. Mechanistically, in the cytoplasm, AKT1 phosphorylates ME2fl at serine 9 in the mitochondrial localization signal peptide at the N-terminus, preventing its mitochondrial translocation. Unlike mitochondrial ME2, which accounts for adjusting the tricarboxylic acid (TCA) cycle, ME2fl functions as a scaffold that brings together the key glycolytic enzymes phosphofructokinase (PFKL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as Lactate dehydrogenase A (LDHA), to promote glycolysis in the cytosol. Thus, through phosphorylation of ME2fl, AKT1 enhances the glycolytic capacity of tumor cells in vitro and in vivo, revealing an unexpected role for subcellular translocation switching of ME2 mediated by AKT1 in the metabolic adaptation of tumor cells to growth stimuli.

Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

How can tracking and tracing systems give us a look at the dark side of the tobacco market?

The aim of this work is to present possible applications of the systems of tobacco traceability for guiding local enforcement against illicit trade. The proposed three-step strategy relies on a robust regression technique and Local Moran's I, a local indicator of spatial association, and aims at identifying retail outlets with significantly low sales compared to normal market conditions, which can indicate illegal sales activities. The ability of the method to produce alerts pointing to areas subject to illicit trade is tested on synthetic data in terms of precision and accuracy in different scenarios. Other metrics are also provided. Our approach performs well under different metrics and across various levels of illicit trade prevalence, achieving a precision of 94% under the main scenario and method parametrisation. The proposed strategy provides high-quality leads for investigations into geographical areas disproportionately susceptible to illicit trade, potentially unveiling any form of illegal sales, including those involving products that have never entered the legal supply chain. Therefore, it can be a valuable tool for law enforcement agencies to tackle illegal sales activities. The findings of this study support also the argument in favour of expanding tobacco traceability systems downstream to the full length of the supply chain.

Recombinant AAV genome size effect on viral vector production, purification, and thermostability

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.