Serum phosphorylated neurofilament heavy-chain levels reflect phenotypic heterogeneity and are an independent predictor of survival in motor neuron disease

Journal of Neurology - To investigate the prognostic role and the major determinants of serum phosphorylated neurofilament heavy -chain (pNfH) concentration across a large cohort of motor neuron...     

Effects of cell penetrating Notch inhibitory peptide conjugated to elastin-like polypeptide on glioblastoma cells

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood–brain barrier penetration. The SynB1–ELP1–dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes’ expression. SynB1–ELP1–dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1–ELP1–dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.     

Structural and enzymatic analyses reveal the binding mode of a novel series of Francisella tularensis enoyl reductase (FabI) inhibitors

Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.     

Magnetic resonance image-guided adaptive stereotactic body radiotherapy for prostate cancer: preliminary results of outcome and toxicity

Objective:Using moderate or ultra-hypofractionation, which is also known as stereotactic body radiotherapy (SBRT) for treatment of localized prostate cancer patients has been increased. We present our preliminary results on the clinical utilization of MRI-guided adaptive radiotherapy (MRgRT) for prostate cancer patients with the workflow, dosimetric parameters, toxicities and prostate-specific antigen (PSA) response.Methods:50 prostate cancer patients treated with ultra-hypofractionation were included in the study. Treatment was performed with intensity-modulated radiation therapy (step and shoot) technique and daily plan adaptation using MRgRT. The SBRT consisted of 36.25 Gy in 5 fractions with a 7.25 Gy fraction size. The time for workflow steps was documented. Patients were followed for the acute and late toxicities and PSA response.Results:The median follow-up for our cohort was 10 months (range between 3 and 29 months). The median age was 73.5 years (range between 50 and 84 years). MRgRT was well tolerated by all patients. Acute genitourinary (GU) toxicity rate of Grade 1 and Grade 2 was 28 and 36%, respectively. Only 6% of patients had acute Grade 1 gastrointestinal (GI) toxicity and there was no Grade ≥ 2 GI toxicity. To date, late Grade 1 GU toxicity was experienced by 24% of patients, 2% of patients experienced Grade 2 GU toxicity and 6% of patients reported Grade 2 GI toxicity. Due to the short follow-up, PSA nadir has not been reached yet in our cohort.Conclusion:In conclusion, MRgRT represents a new method for delivering SBRT with markerless soft tissue visualization, online adaptive planning and real-time tracking. Our study suggests that ultra-hypofractionation has an acceptable acute and very low late toxicity profile.Advances in knowledge:MRgRT represents a new markerless method for delivering SBRT for localized prostate cancer providing online adaptive planning and real-time tracking and acute and late toxicity profile is acceptable.     

The Content of Cobalt,Silver and Vanadium in Raw Cow’s Milk in Croatia and Risk Assessment for Consumers

Concentrations of selected trace elements Ag, Co and V in raw milk sampled from four geographical regions in Croatia were measured. Silver, Co and V were detected above the limit of detection within the range of 9.52%–30.8%, 1.6%–12.1% and 12.4%–30.8%. Silver concentrations were not detected in milk samples from the Croatian Littoral and Mountainous Croatia (CL-MC) region. Similar Ag content was found in Southern, Eastern and Central Croatia. The lowest mean of Co and V of 33.2 and 83.8 µg kg^?1 were found in the CL-MC region while the highest of 49.8 and 136.9 µg kg^?1 was found in Central Croatia. There were no statistically significant differences in Ag, Co and V contents between the four regions. The estimated daily dietary intakes (EDI) of total mean and total 95th percentile values of Ag, Co and V showed lower values in comparison with available EFSA health-based limits.

     

Cell membrane-related toxic responses and disruption of intercellular communication in PCB mechanisms of toxicity: A review

An understanding of polychlorinated biphenyl (PCB) congener-specific effects on cell membrane and intercellular communication is important within the studies of PCB absorption, organ-related PCB accumulation and exertion of toxic responses. Toxic potential of PCBs is linked to various deleterious effects on human health, including neurotoxicity, immunotoxicity, reproductive toxicity and genotoxicity and, recently in 2016 International Agency for Research on Cancer (IARC) has upgraded the classification of PCBs to Group 1 “Carcinogenic to humans.” Proposed mechanisms of aforementioned PCBs adverse effects at cellular membrane level are: (i) downregulation of gap junction intercellular communication and/or connexins; (ii) compromised membrane integrity; and (iii) altered tight junction barrier function. This study, based on an extensive literature survey, shows the progress in scientific research of each of these three levels with the aim of pointing out the earliest toxic events of PCBs, which can result in serious cell/tissue/organ damage.     

Improved Anti‐Emetic Efficacy of 5‐HT3 Receptor Antagonists in Cancer Patients with Genetic Polymorphisms of ABCB1 (MDR1) Drug Transporter

Chemotherapy‐induced nausea and vomiting (CINV) remains a major adverse effect decreasing quality of life in patients with cancer. Genetic variations among patients may be responsible for part of the lack of efficacy of anti‐emetic drugs. The aim of this study was to investigate how the genetic variants of the drug transporter ABCB1 (MDR1) gene affect anti‐emetic treatment with 5‐HT₃ receptor antagonists. Patients (n = 239) receiving moderately or highly emetogenic chemotherapy and ondansetron or granisetron were included in the study. Anti‐emetic responses were recorded daily. The primary end‐point of the assessment was the total control rates of CINV in the acute and delayed phases after chemotherapy. Genotyping was performed by PCR‐RFLP. In the acute phase, patients with ABCB13435TT, 1236TT or 2677TT genotypes had a higher control rate of CINV than other genotype groups: (64.7% in 3435TT versus 45.7% in 3435CC+CT, p = 0.016; 65.1% in 1236TT versus 46.4% in 1236CC+CT, p = 0.027; 66.7% in 2677TT versus 46.5% in other genotypes, p = 0.021). Subjects carrying homozygous variant alleles together (TT‐TT‐TT) showed a significantly higher protection from nausea and vomiting (67.7% in TT‐TT‐TT versus 47.1% in other genotypes, p = 0.032). After the logistic regression analysis with adjustment for other known covariates, the total control rate was significantly higher in the 3435TT genotype group during the acute phase (p = 0.021). No significant change was found between the total control rates among genotypes in the delayed phase. Each of three 3435TT, C1236TT, 2677TT genotypes of ABCB1 and their combination was associated with about 50% higher anti‐emetic response to 5‐HT₃ receptor antagonists in the acute phase of chemotherapy in patients with cancer receiving moderately or highly emetogenic chemotherapy. ABCB1 (MDR1) genotypes may contribute to predict the anti‐emetic efficacy of 5‐HT₃ antagonists.     

Mechanism and kinetics of a sodium-driven bacterial flagellar motor

The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque–speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque–speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a “powerstroke” in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration.Ion-motive force (IMF) comprises electrical and chemical transmembrane potentials and is defined aswhere V_m is the membrane voltage and Δμ = k_BT ln(C_in/C_out) and q are the transmembrane chemical potential difference and charge of the ions, respectively, with C_in and C_out the internal and external ion concentrations. In most species the primary form of biological free energy is the proton-motive force (PMF), the IMF for H⁺ ions (1). Physiological PMF is typically in the range −150 mV to −200 mV, with the inside electrically negative and slightly alkaline relative to the outside. Some organisms use sodium-motive force (SMF) to drive numerous cellular processes, such as bacterial motility (2), ATP synthesis (3), and active membrane transport (4). Arguably the most important process driven by IMF is ATP synthesis, which generates cellular ATP by forced rotation of the F₁ part of F₁F_O ATP-synthase. F₁ is mechanically coupled to and rotated by F_O, which like the bacterial flagellar motor (BFM) is an ion-driven rotary motor. Understanding the mechanism of these and other ion-driven molecular machines is a fundamental challenge in cellular energetics and biophysics.The BFM (Fig. 1A) is a rotary molecular machine that propels many species of swimming bacteria. It couples ion flow, for example protons (H⁺) in Escherichia coli or sodium ions (Na⁺) in Vibrio alginolyticus, to the rotation of extracellular helical flagellar filaments at hundreds of revolutions per second (Hz) (2, 5, 6). Torque is generated by interactions between stator complexes (containing the proteins MotA and MotB in E. coli and PomA and PomB in V. alginolyticus) and the rotor protein FliG (7). In E. coli, each motor can be powered by any number between 1 and at least 11 functionally independent stators (8), which exchange with a membrane-bound pool of “spare” stators on a timescale of minutes (9).Open in a separate windowFig. 1.Bacterial flagellar motor speed measurement. (A) Schematic of the bacterial flagellar motor and the polystyrene bead rotation assay. Stator proteins couple ion flux to rotation of the rotor. The hook is a universal joint that connects external flagellar filaments to the rotor. Motor rotation is observed via back-focal-plane interferometry of a polystyrene bead attached to the truncated filament. (B) A typical 10-ms X-Y trace of a 200-nm polystyrene bead attached to a fully energized motor, sampled at 10 kHz. (C) The power spectrum of (X + iY) from B shows a clear peak at 660 Hz. (D) The stable rotational speed of the same motor for 10 s.The most important biophysical method for studying the torque-generating mechanism of the BFM has been to measure its torque–speed curves. This has been done using varying viscous load (10–14) or external torque (15, 16) to control the speed. Fully energized motors with a full complement of stators, driven by H⁺ (10) or Na⁺ (11, 12), show the same characteristic torque–speed curve. There is a plateau of nearly constant torque from stall up to a certain limiting speed, and at higher speeds torque falls more rapidly to zero. The junction between these two domains is relatively sharp and has been called the “knee”. In the wild-type E. coli motor, the knee is at ∼200 Hz and the zero-torque speed is ∼300 Hz (17). Na⁺-driven motors are faster: In V. alginolyticus and the chimeric motor in E. coli the knee speeds are ∼450 Hz and the zero-torque speeds are ∼700 Hz and ∼900 Hz, respectively (11, 12). Torque–speed curves have also been measured for fully energized motors with reduced numbers of stators. The torque generated by H⁺-driven motors with N stators appears to be simply N times the torque generated by a single stator at the same speed (18), a result that has recently been confirmed by measurements close to the zero-torque limit (17, 19). Previously measured torque–speed curves for single-stator Na⁺-driven motors do not extend beyond the knee due to a lack of data points at extremely low load (12). Various models of the mechanism of torque generation in the BFM have been proposed and tested by their ability to predict the torque–speed curve (16, 20–24). The torque plateau is believed to represent a regime where motion of the attached load rather than internal processes in the motor’s mechano-electrochemical cycle is rate limiting, and the system operates with high efficiency close to thermodynamic equilibrium. Thus, the plateau torque is close to the stall torque, and the work done against viscous drag is nearly equal to the free energy of driving ions that is consumed by the motor. As the load is reduced and the motor speeds up, the knee represents the point at which internal processes become rate limiting, and the zero-torque speed is a measure of the limiting rate of the cycle in the absence of load.The recent development of a Na⁺-driven chimeric flagellar motor in E. coli (25) opened new possibilities for investigation of the mechano-electrochemical cycle of the BFM (26). The PMF that drives the wild-type motor of E. coli is the primary form of free energy in the cell and is consequently tightly regulated and relatively difficult to manipulate. Furthermore, extreme changes in H⁺ concentration equate to extremes of pH, which are expected to affect the stability of motor proteins. On the other hand, the SMF in E. coli is secondary and relatively easy to manipulate, and extremes of [Na⁺] are tolerated by the cell. We have recently demonstrated that the electrical and chemical potential components of the SMF in E. coli can be varied independently via the pH and [Na⁺] of the surrounding medium (27, 28) (Table S1).In this paper, we measured the torque–speed curves of single-stator Na⁺-driven chimeric motors for each of 25 different combinations of membrane voltage and sodium gradient (27, 28) (Table S1). We chose single-stator motors, to isolate the properties of the fundamental mechanochemical cycle from the complication of interactions between different stators in the same motor. Our set of torque–speed curves is an order of magnitude bigger than any previous torque–speed dataset and allowed a systematic exploration of the parameter space of a minimal four-state kinetic model of the BFM mechanochemical cycle. The four-state kinetic model derives from the simplest possible representation of a carrier-like ion transport mechanism (29, 30) and has been extensively used to model the BFM (31). Rather than choosing a single set of model parameters by educated guesswork, as in all previous attempts to model the flagellar motor, we performed a comprehensive search for sets of values of the 25 model parameters that were consistent with our measured torque–speed curves.     

Time Analysis of Online Adaptive Magnetic Resonance–Guided Radiation Therapy Workflow According to Anatomical Sites

PurposeTo document time analysis of detailed workflow steps for the online adaptive magnetic resonance–guided radiation therapy treatments (MRgRT) with the ViewRay MRIdian system and to identify the barriers to and solutions for shorter treatment times.Methods and MaterialsA total of 154 patients were treated with the ViewRay MRIdian system between September 2018 and October 2019. The time process of MRgRT workflow steps of 962 fractions for 166 treatment sites was analyzed in terms of patient and online adaptive treatment (ART) characteristics.ResultsOverall, 774 of 962 fractions were treated with online ART, and 83.2% of adaptive fractions were completed in less than 60 minutes. Sixty-three percent, 50.3%, and 4.2% of fractions were completed in less than 50 minutes, 45 minutes, and 30 minutes, respectively. Eight-point-three percent and 3% of fractions were completed in more than 70 minutes and 80 minutes, respectively. The median time (t_med) for ART workflow steps were as follows: (1) setup t_med: 5.0 minutes, (2) low-resolution scanning t_med: 1 minute, (3) high-resolution scanning t_med: 3 minutes, (4) online contouring t_med: 9 minutes, (5) reoptimization with online quality assurance t_med: 5 minutes, (6) real targeting t_med: 3 minutes, (7) beam delivery with gating t_med: 17 minutes, and (8) net total treatment time t_med: 45 minutes. The shortest and longest t_mean rates of net total treatment time were 41.59 minutes and 64.43 minutes for upper-lung-lobe-located thoracic tumors and ultracentrally located thoracic tumors, respectively.ConclusionsTo our knowledge, this is the first broad treatment-time analysis for online ART in the literature. Although treatment times are long due to human- and technology-related limitations, benefits offered by MRgRT might be clinically important. In the future, implementation of artificial intelligence segmentation, an increase in dose rate, and faster multileaf collimator and gantry speeds may lead to achieving shorter MRgRT treatments.