High Inhibitory Concentration Research Articles

Towards the Targeted Protein Degradation of PRMT1.

Targeting the protein arginine methyltransferase 1 (PRMT1) has emerged as a promising therapeutic strategy in cancer treatment. The phase 1 clinical trial for GSK3368715, the first PRMT1 inhibitor to enter the clinic, was terminated early due to a lack of clinical efficacy, extensive treatment-emergent effects, and dose-limiting toxicities. The incidence of the latter two events may be associated with inhibition-driven pharmacology as a high and sustained concentration of inhibitor is required for therapeutic effect. The degradation of PRMT1 using a proteolysis targeting chimera (PROTAC) may be superior to inhibition as proceeds via event-driven pharmacology where a PROTAC acts catalytically at a low dose. PROTACs containing the same pharmacophore as GSK3368715, combined with a motif that recruits the VHL or CRBN E3-ligase, were synthesised. Suitable cell permeability and target engagement were shown for selected candidates by the detection of downstream effects of PRMT1 inhibition and by a NanoBRET assay for E3-ligase binding, however the candidates did not induce PRMT1 degradation. This paper is the first reported investigation of PRMT1 for targeted protein degradation and provides hypotheses and insights to assist the design of PROTACs for PRMT1 and other novel target proteins.

Autohydrolysis of sugarcane bagasse with water reuse: Impacts on residues’ composition and enzymatic hydrolysis

This work presents a new sequential approach to the sugarcane bagasse autohydrolysis process in a way that the liquor from the previous reaction was reused in the next one, and the makeup water for the next batch was used to wash the solid fraction before being added to the liquor in the next batch. This approach was suggested first as a way of reducing the water usage in the process, and second as a way of concentrating the liquor in any interesting component, working with the possibility of losing efficiency towards the glucose production through a cellulose loss to the liquor or enzymatic efficiency loss from a higher inhibitor concentration. Two sets of five sequential batches were performed: one washing the solids with the makeup water prior to the enzymatic step (Set 2), and the other one without the washing step (Set 1), looking forward to the effects of the water reuse over the glucose production and the liquor composition. Autohydrolysis pretreatment removed most of the hemicellulose (∼94 %), with liquor recycling improving its removal until the third batch and stabilizing after that. Although the washing step showed little impact on the composition of the solids, it was determinant to the success of the enzymatic hydrolysis, since it was possible to maintain the cellulose to glucose yield around 53% throughout the batches. There was a 64 % reduction in the water used in the sequential reactions without interfering in the glucose production, which indicates that the proposed strategy could be successfully used to reduce costs and environmental impacts associated with the pretreatment of lignocellulosic biomass.

Assessment of Common Factors Associated with Droplet Digital PCR (ddPCR) Quantification of Paratrichodorus allius in Soil.

This research investigated the factors associated with the quantitative detection of Paratrichodorus allius in soil using droplet digital PCR (ddPCR). Small-sized nematodes exhibited significantly lower DNA quantities compared to their medium and large counterparts. Soil pre-treatments (room temperature drying and 37 °C oven-drying) demonstrated no substantial impact on ddPCR detection, and soil storage (0-3 months at 4 °C) exhibited negligible alterations in DNA quantities. A commercial DNA purification kit improved the resulting quality of ddPCR, albeit at the cost of a notable reduction in DNA quantity. Upon assessing the impact of inhibitors from soil extracts, a higher inhibitor concentration (5%) influenced ddPCR amplification efficiency. Incorporating bovine serum albumin (BSA) (0.2 μg/μL or 0.4 μg/μL) into the ddPCR setup mitigated the issue. In brief, while ddPCR exhibits minimal sensitivity to soil pre-treatments and storage, higher concentrations of PCR inhibitors and the DNA purification process can influence the results. Despite ddPCR's capability to detect nematodes of all sizes, quantification may not precisely reflect soil population. Incorporating BSA into the ddPCR setup enhances both detection and quantification capacities. This study represents the first comprehensive investigation of its kind for plant-parasitic nematodes, providing crucial insights for application of ddPCR in nematode diagnosis directly from the soil DNA.

Studying the Effectiveness of an Expired Betamethasone Drug in Sulfuric Acid Solutions to Examine the Corrosive Behavior of Copper Using Weight Loss and Experimental Design

Utilizing expired pharmaceuticals as corrosion inhibitors for copper in acidic environments offers compelling advantages, including cost-effectiveness, reduced toxicity compared to traditional inhibitors, and contribution to pharmaceutical waste reduction through recycling. This study investigates the corrosion inhibition of copper in a sulfuric acid solution using varying concentrations of Expired Betamethasone Drug, employing weight loss and Experimental Design methods. The influence of temperature on copper's corrosion behavior is examined within the range of 293–333 K. Results show that inhibition efficiency increases with higher inhibitor concentrations but decreases with rising temperature. Thermodynamic analyses elucidate adsorption and activation processes, revealing that the adsorption of Expired Betamethasone Drug on copper surfaces is characterized as endothermic and spontaneous, aligning well with the Langmuir and Frumkin adsorption isotherms. The activation and free energies of inhibition reactions support a mechanism of physical adsorption. To establish the relationship between factors and responses, we employ response surface methodology (RSM) with regression statistical analysis and probabilistic assessment. Statistical analysis demonstrates highly significant quadratic models for inhibition efficiencies (IE) with a coefficient of multiple regressions (R²) of 0.999. Further model validation confirms a strong fit (adjusted R² = 0.997), with experimental observations closely matching predictions and a highly significant model (Q² = 0.989). The findings reveal that this expired drug exhibits substantial inhibitory power, exceeding 96%, in both experimental and predictive calculations.

Isolation and Characterization of Enterobacter sp Capable towards Tolerating Degradation Products and Fermenting Pentoses

<p>The depletion of fossil fuel resources, in addition to the growing demand for energy, has prompted the development of renewable energies, including bioethanol from lignocellulosic biomass. The acid pretreatment of hemicellulose releases inhibiting compounds in addition to xylose. With the possibility of exploitation of lignocellulose as a fermentation substrate, we isolated an <em>Enterobacter </em>characterized by its ability to ferment xylose and tolerate high concentrations of inhibitors. The selection was performed in media containing different carbon and energy sources; glucose, cellobiose, CMC, furfural and 5-HMF. Characterization strategies of the selected strain such as, xylose concentration (from 25 g liter-1 to 100 g liter-1), furfural (0 mM to 25 mM), cell immobilization, were used to quantify the maximum yield of ethanol produced. The results obtained show that our strain can ferment up to 100 g liter-1 of xylose in the presence of 20 mM furfural at 37°C to produce ethanol with a maximum yield of 2.22 g liter- 1 for 24 h under 160 rpm magnetic stirring. The results obtained in this study suggest that the isolated <em>Enterobacter </em>sp. is a promising strain for the bioconversion of lignocellulosic biomass pretreatment hydrolysate into bioethanol.</p><p> </p>

Anticorrosion studies of 5-acetyl-4-(3-methoxyphenyl)-6-methyl-1-phenyl-3,4-dihydropyrimidin-2(1H)-one: approach from experimental, DFT studies, and MD simulation

Abstract The effectiveness of 5-acetyl-4-(3-methoxyphenyl)-6-methyl-1-phenyl-3,4-dihydropyrimidin-2(1H)-one as a corrosion inhibitor for mild steel in acidic conditions was investigated herein through the experimental and theoretical approach. Experimental results demonstrated that this compound acts as a reliable corrosion inhibitor (η %) for mild steel in acidic environments, with its inhibition efficiency increasing as the inhibitor concentration rises. Adsorption behavior on the mild steel surface followed Langmuir and Temkin adsorption isotherms. Electrochemical polarization tests indicated that the compound exhibited a mixed corrosion type, and impedance spectroscopy revealed an increase in charge transfer resistance with higher inhibitor concentrations. Examination of the mild steel surface using SEM and Atomic Force Microscopy (AFM) confirmed the formation of a protective film. Wettability characteristics were assessed using the contact angle method. Frontier molecular orbital analysis revealed the HOMO and LUMO values for both the neutral and protonated forms of the compound. At 289 °C, the interaction energy for adsorption was found to be approximately −146.3006 kJ/mol for the neutral system and −135.8122 kJ/mol for the protonated system, while at 318 °C, the corresponding values were −140.6106 kJ/mol and −147.6022 kJ/mol. These findings collectively suggest the potential industrial utility of the investigated inhibitor as an effective corrosion inhibitor.

Abstract A037: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer

Abstract Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. However, tumors harboring EGFR exon 20 insertions have been historically more difficult to target. Agents such as amivantamab and mobocertinib have recently been granted accelerated approval for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation by inhibitor treatment of sensitive cell lines, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. Incubation with a high concentration of inhibitor selected for drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT (epithelial-mesenchymal transition). Conversely, gradual escalation of inhibitor concentrations resulted in acquisition of oncogenic NRAS mutations. In a parallel approach, we performed a deep mutational scanning assay of the EGFR D770_N771insSVD kinase domain, replacing each amino acid with every other possible amino acid, identifying both known and novel mutations that cause resistance to BAY 2927088 and other EGFR inhibitors. As expected, the C797S mutation was enriched as a mechanism of acquired resistance only to irreversible EGFR exon 20 insertion inhibitors. Surprisingly, the T790M gatekeeper mutation was found to cause resistance to all EGFR exon 20 insertion inhibitors tested, including irreversible inhibitors. Several novel resistance mutations clustering predominantly in exons 20 and 21 were also identified. Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Kristen Doucette, Hasmik Keshishian, Steven Carr, Xiaoping Yang, David Root, Andrew D Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A037.

Detection of the effect of silver nanoparticles prepared by using extract leaves Prosperis juliflora on pathogenic fungus Trichophyton tonsurans isolated from human scalp

The silver nanoparticles were prepared using Prosopis juliflora leaf extract and extracted from trees in Kirkuk city and showed that the silver nitrate solution in 1 mm turned its color brown which will be preliminary evidence of the formation of nanoparticles. The UV and visible absorption spectrum of a solution of silver nanoparticles was tested as a second step in confirming the formation of the particles, and it was found that they were present at the wavelength (440) nm, where it turned out that the peaks of X-ray diffraction were at (111), (220), (311) at angles (31.77, 48.11, 54.21) respectively. Trichophyton tonsurans were isolated from the scalp of a person with ringworm from Kirkuk General Hospital. The fungus was diagnosed on the basis of the phenotypic and microscopic characteristics and hair penetration. it was found that the solution of silver nanoparticle had a clear inhibitory effect on the fungus Trichophyton tonsurans. The highest and lowest inhibitor concentration reached 90% and the highest inhibitor concentration was 100 % Also, the ratios of the silver nanoparticles effects on the growth and production of fungi spores were determined.

Mild steel corrosion inhibition comparison between indole-5-carboxylic acid and benzofuran-5-carboxylic acid: An integrated experimental and theoretical study

Corrosion inhibition capabilities of Indole-5-carboxylic acid (IC) and Benzofuran-5-carboxylic acid (BC) on mild steel have been comprehensively investigated using various techniques, including Potentiodynamic Polarization Measurement (PPM), electrochemical impedance spectroscopy (EIS), and scanning electrochemical microscopy (SEM). The results from polarization tests demonstrated that both IC and BC effectively reduced the corrosion current densities of both cathodic and anodic reactions occurring on the metal surface. Through EIS analysis, it was observed that the inhibitory efficacy of IC and BC increased with higher inhibitor concentrations. At a temperature of 30°C, the solution containing 10−2M IC and 10−2M BC exhibited the maximum inhibitory efficiency, with IC achieving 92.19% and BC achieving 75.00%. The adsorption behavior of the inhibitors was found to follow the Langmuir isotherm, suggesting a monolayer adsorption process. Additionally, quantum chemical calculations were carried out to assess the molecular parameters of the inhibitors thoroughly. These calculations consistently indicated that IC exhibited superior inhibitory properties compared to BC. Furthermore, molecular dynamics simulations were employed to gain insights into the optimal adsorption configuration of the inhibitors on the Fe(110) surface. The results revealed that IC possessed a higher binding energy with the Fe(110) surface, confirming its superior ability to inhibit steel corrosion compared to BC.

ERG K+ channels mediate a major component of action potential repolarization in lymphatic muscle

Smooth muscle cells in the walls of collecting lymphatic vessels fire spontaneous action potentials (APs), which conduct rapidly over the muscle layer to initiate contractions that propel lymph. Several ion channels have been implicated in the currents underlying the AP spike and the preceding diastolic depolarization, but the molecular identities of K+ channels involved in AP repolarization are unknown. Based on previous studies of other rhythmically active smooth muscles, we hypothesized that ether-a-go-go related gene (ERG) K+ channels (Kv11) play an important role in repolarization of the AP in lymphatic muscle. Message for one or more ERG channel isoforms was detected by RT-PCR analysis of lymphatic vessels from mice, rats and humans. Membrane potential recordings in smooth muscle cells of rat and human lymphatics revealed that nanomolar concentrations of ERG-1 inhibitors (E-4031 and BeKm-1) prolonged the duration of the AP plateau (normally ~ 1 s in duration) and induced multiple spikes, whereas ERG-1 activators (ICA-105574 and RPR-260243) shortened the plateau and could completely inhibit spontaneous APs. At relatively high inhibitor concentrations, the AP plateau duration lasted as long as 24 s. ERG activators reversed the effects of ERG inhibitors and vice-versa. In pressure myograph studies, ERG channel inhibition prolonged the diastolic repolarization phase of the contraction cycle and reduced the frequency of spontaneous contractions. This is the first evidence for a specific K+ channel contributing to the AP in lymphatic muscle. Our results imply that lymphatic contractile dysfunction may occur in long QT type II patients with mutations that result in ERG channel loss-of-function or impaired trafficking of the channel to the cell membrane.

Investigating the use of Moringa Oleifera leaf extract as an environment-friendly corrosion inhibitor for API 5L X52 steel in 1 M HCl

This work studied the potential of using moringa oleifera leaf extract (MOLE) as an eco-friendly inhibitor for API 5L X52 steel in acidic media at 0.1 g/L–0.5 g/L concentration and 25 °C–60 °C. The study also evaluated the impact of adding potassium iodide (KI) to the corrosion-inhibiting properties of MOLE. Results from electrochemical testing showed that MOLE exhibited a high inhibition efficacy of 99% at 0.5 g/L concentration and 60 °C. The findings indicated that MOLE functions as a mixed-type corrosion inhibitor. Its efficiency increases with higher inhibitor concentrations following a Langmuir adsorption isotherm, and it is physically adsorbed on the steel surface. FT-IR spectra analysis revealed the presence of hydroxyl, carbonyl, saturated, and unsaturated aliphatic functionalities in the MOLE. The decreased absorption intensity in UV–vis spectra after 2 and 4 h of corrosive acid environment exposure indicates that MOLE was well adsorbed onto the metal surface. Hence, adding KI to the MOLE-containing samples enhanced the inhibitor's adsorption. The SEM-EDX observations supported the findings from electrochemical characterization and confirmed MOLE’s high inhibition performance. Hence, this study suggests that MOLE has promising potential as a green corrosion inhibitor for the API 5L X52 steel in acidic media.

Investigation of the corrosion inhibition performance of snail slime on the exposed surface of mild steel in acidic environment

Corrosion of metals is a major issue in various industries and the use of organic materials, especially plant extracts, as alternatives to toxic chemicals in corrosion inhibitors is gaining attention. However, the potential of animal products as corrosion inhibitors has not been fully explored. This study investigates the corrosion inhibiting potential of snail slime (SS) for mild steel in 0.5 M H2SO4 solution using Gravimetric and electrochemical methods, including open circuit potential (OCP), potentiodynamic polarization (PDP), and electrochemical impedance spectroscopy (EIS). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were used for material characterization. The results indicate that snail slime has a high inhibition efficiency of up to 92.75%, with inhibition efficiency decreasing as temperature and immersion time increase but it increased with higher inhibitor concentrations. Adsorption studies showed that snail slime exhibited strong adherence to the Langmuir and Temkin adsorption isotherms, indicating spontaneous adsorption on mild steel. The inhibition mechanism shows typical physical adsorption and follows first-order kinetics. While the EIS analysis indicated a charge transfer-controlled corrosion process with the highest inhibition efficiency of 81.74%, the PDP analysis revealed that snail slime acted as a mixed-type inhibitor with the highest inhibition efficiency of 81.98%. FTIR reveals the functional groups responsible for the inhibition exhibited by snail slime inhibitors, including CH, OH, CO-O, CCl, and NH. SEM shows that the inhibition of corrosion is due to the formation of an insoluble stable protective film on the sample surface by an adsorption process. These findings suggest that snail slime has significant potential as an eco-friendly alternative to synthetic corrosion inhibitors in industrial applications.

Biosensor based on Cellulose Acetate/Glutaraldehyde Membrane Electrodes for detection of organophosphorus pesticides

In recent years, sensor applications have been critical in many fields, especially food safety and pesticides. Organophosphorus pesticides (OPPs) can be detected using a potentiometric biosensor with a membrane electrode made of a new natural material based on cellulose acetate (CA). Acetylcholinesterase was immobilized to 15% modified CA membrane electrodes using glutaraldehyde (GTA) as crosslinking agent and gold (Au) electrode. An indirect method used an acetylthiocholine chloride (ATCl) substrate to find OPPs like chlorpyrifos, profenophos, and diazinon. The working electrode was an CA/GTA membrane electrode, and the reference electrode was an Ag/AgCl electrode, whose potential value was measured with a potentiometer. The surface morphology of the biosensor membrane was investigated using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX). It showed that the CA membrane has a smooth, porous surface and is very dense, and its structure consists of 71.27% carbon (C) and 28.73% oxygen (O) with an average diameter of 562.33 nm. A potentiometric biosensor based on AChE inhibition for the detection of OPPs showed a limit of detection (LoD) of 1×10−6 μg/L with a linearity range of 1×10−6–1.0 μg/L. The %inhibition value for the chlorpyrifos pesticide was 14.44 to 73.08%, profenophos was 11.98 to 77.98%, and diazinon was 18.58 to 83.27%. Therefore, higher inhibitor concentrations (OPPs) have a greater ability to prevent the AChE enzyme from breaking down the acetylcholine substrate. The biosensor with the CA membrane has a wide linearity range and a low detection limit. The potentiometer rapidly detects pesticide residues.

Detoxification of high solid-liquid hydrothermal pretreated sugar cane bagasse by chromatographic adsorption for cellulosic ethanol production

The conversion of sugar cane bagasse (SCB) into cellulosic ethanol by microorganism becomes challenging at high solid liquid ratio (SLR) due to high concentration of inhibitors in the liquid hot water pretreatment. Therefore, this study aims to produce cellulosic ethanol by detoxifying the high SLR hydrolysate (1:6, w/v) using SY-01 resin. The detoxification effects of SY-01 resin dosage and adsorption modes were systematically investigated. The resulting detoxified hydrolysate was successfully fermented, and the highest ethanol concentration achieved was 30.94 ± 0.13 g/L. Additionally, the ethanol yield was 102.51 ± 0.94%, and the ethanol production from SCB was 210 ± 1 mg/g SCB. Our findings confirm that SY-01 resin is effective in detoxifying lignocellulosic enzymatic hydrolysate, providing an economical and feasible method for the high-value utilization of biomass resources.

Abstract 433: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer

Abstract Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. Tumors harboring EGFR exon 20 insertions do not respond to inhibitors of classical activating mutations. Agents such as amivantamab and mobocertinib have been approved for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. With the first method, involving initial incubation with a high concentration of inhibitor, we isolated drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT. With the second method, involving gradual escalation of inhibitor concentrations, we identified NRAS hotspot mutations as a mechanism of resistance to BAY 2927088 and other EGFR inhibitors. As a parallel approach, we are developing a deep-scanning mutagenesis assay of the EGFR kinase domain to identify mutations that could cause resistance. Understanding these resistance mechanisms will help to identify second-line treatments for exon 20 insertion patients who develop resistance to EGFR inhibition. Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Andrew Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 433.

Modulatory effect of MG-132 proteasomal inhibition on boar sperm motility during in vitro capacitation.

A series of biochemical and biophysical changes during sperm capacitation initiates various signaling pathways related to protein phosphorylation leading to sperm hyperactivation, simultaneously with the regulation of proteasomal activity responsible for protein degradation and turnover. Our study aimed to unveil the role of the proteasome in the regulation of boar sperm motility, hyperactivated status, tyrosine phosphorylation, and total protein ubiquitination. The proteolytic activity of the 20S proteasomal core was inhibited by MG-132 in concentrations of 10, 25, 50, and 100 μM; and monitored parameters were analyzed every hour during 3 h of in vitro capacitation (IVC). Sperm motility and kinematic parameters were analyzed by Computer Assisted Sperm Analysis (CASA) during IVC, showing a significant, negative, dose-dependent effect of MG-132 on total and progressive sperm motility (TMOT, PMOT, respectively). Furthermore, proteasomal inhibition by 50 and 100 μM MG-132 had a negative impact on velocity-based kinematic sperm parameters (VSL, VAP, and VCL). Parameters related to the progressivity of sperm movement (LIN, STR) and ALH were the most affected by the highest inhibitor concentration (100 μM). Cluster analysis revealed that the strongest proteasome-inhibiting treatment had a significant effect (p ≤ 0.05) on the hyperactivated sperm subpopulation. The flow cytometric viability results proved that reduced TMOT and PMOT were not caused by disruption of the integrity of the plasma membrane. Neither the protein tyrosine phosphorylation profile changes nor the accumulation of protein ubiquitination was observed during the course of capacitation under proteasome inhibition. In conclusion, inhibition of the proteasome reduced the ability of spermatozoa to undergo hyperactivation; however, there was no significant effect on the level of protein tyrosine phosphorylation and accumulation of ubiquitinated proteins. These effects might be due to the presence of compensatory mechanisms or the alteration of various ubiquitin-proteasome system-regulated pathways.

FEA study on the TSV copper filling influenced by the additives and electroplating process

Through-Silicon Via (TSV) is a key technology of three-dimensional (3D) integration. However, Voids are usually found in the Cu filling process of TSV, and lead to lower reliability. Additives and optimized filling process are used to solve this problem. However, the comprehensive analysis of these affecting parameter needs many experiments and cost much time. Thus, the finite element analysis (FEA) for the mass transferring of electroplating is carried out by the COMSOL software in the work. Different inhibitor concentrations in filling process are first studied and the most suitable density of 64 mol/m2 is obtained. However, high inhibitor concentration will lead to much lower plating speed. Thus, the effect of filling process such as current density and plating bath flow velocity on the voids are investigated under the optimized concentrations above. The best filling quality is obtained at the inhibitor concentration of 64 mol/m2, current density of 1.4 ASD and the bath stirring velocity of 0.4 m/s. The underlying growth mechanism of these influencing factors are also analyzed and revealed.

Inhibition of Starch Digestion by α-Amylase Inhibitor Reduces the Efficiency of Utilization of Dietary Proteins and Lipids and Retards the Growth of Rats

Digestion/absorption and nutritional utilization of starch, protein and lipids were studied in rats fed diets containing purified kidney bean α-amylase inhibitor at levels of 0, 1.6, 3.3 and 6.6 g/kg diet. At the two higher levels, the growth rate of rats and the apparent digestibilities and utilization of dietary starch and protein were significantly less than in control rats, and losses of nitrogen, lipids and carbohydrate resulted in a significant reduction in dry body weight. Some organs of the body were also affected: the relative dry weights of the intestines and the pancreas were higher, whereas liver and thymus weights were lower than in control rats. As starch digestion in the small intestine was negligible at higher inhibitor concentrations, the cecum was practically blocked by solidified digesta. This effect and the ensuing bacterial fermentation stimulated the growth of this tissue by hyperplasia and hypertrophy. However, as the distension was not always sufficient, the organ was occasionally ruptured and the rats had to be killed. Inhibitor doses in this work were comparable to those in clinical studies, implying that the use of the inhibitor is not without health risks. Moreover, diets rich in α-amylase inhibitor such as those containing transgenic plants with high levels of inhibitor gene expression cannot be recommended in intensive animal production.

Macrophage colony-stimulating factor potentially induces recruitment and maturation of macrophages in recurrent pituitary neuroendocrine tumors.

Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.

Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.