Intracellular Fraction Research Articles

Glycolate production by a Chlamydomonas reinhardtii mutant lacking carbon-concentrating mechanism

The green alga Chlamydomonas reinhardtii serves as a model organism for plant and photosynthesis research due to many commonalities in metabolism and to the fast growth rate of C. reinhardtii which accelerates experimental turnaround time. In addition, C. reinhardtii is a focus of research efforts in metabolic engineering and synthetic biology for the potential production of biofuels and value-added chemicals. Here, we report that the C. reinhardtii cia5 mutant, which lacks a functional carbon-concentrating mechanism (CCM), can produce substantial amounts of glycolate, a high-value cosmetic ingredient, when the mutant is cultured under ambient air conditions. In order to reveal the metabolic basis of glycolate accumulation by the cia5 mutant, we investigated the metabolomes of the cia5 mutant and a wild type strain CC-125 (WT) through the global metabolic profiling of intracellular and extracellular fractions using gas chromatography and mass spectrometry. We observed the intracellular and extracellular metabolic profiles of the WT and the cia5 mutant were similar during the mixotrophic phase at 30 h. However, when the cells entered the photoautotrophic phase (i.e., 96 h and 120 h), both the intracellular and extracellular metabolic profiles of cia5 mutant differed significantly when compared to WT. In the cia5 mutant strain, a group of photorespiration pathway intermediates including glycolate, glyoxylate, glycine, and serine accumulated to significantly higher levels compared to WT. In the photorespiration pathway, glycolate is metabolized to glyoxylate and glycine leading to NH3 and CO2 generation during the mitochondrial conversion of glycine to serine. This result provides further evidence that the CIA5 mutation increased the photorespiration rate. Because the cia5 mutant lacks a CCM, and C. reinhardtii might harbor an inefficient or incomplete photorespiration pathway, glycolate may accumulate when the CCM is not functional. We envision that investigating photorespiration controls in C. reinhardtii provides tools for producers to use the cia5 mutant to produce glycolate as well as platform to engineer alternative pathways for glycolate metabolism.

Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B 1 elimination ability

The use of microorganisms for Aflatoxin B1 elimination has been studied as a new alternative tool and it is known that cell wall carried out a critical role. For that reason, cell wall and soluble intracellular fraction of eight yeasts with AFB1 detoxification capability were analysed. The quantitative and qualitative comparative label-free proteomic allowed the identification of diverse common constituent proteins, which revealed that putative cell wall proteins entailed less than 10% of the total proteome. It was possible to characterize different enzymes linked to cell wall polysaccharides biosynthesis as well as other proteins related with the cell wall organization and regulation. Additionally, the concentration of the principal polysaccharides was determined which permitted us to observe that β-glucans concentration was higher than mannans in most of the samples. In order to better understand the biosorption role of the cell wall against the AFB1 , an antimycotic (Caspofungin) was used to damage the cell wall structure. This assay allowed the observation of an effect on the normal growth of those yeasts with damaged cell walls that were exposed to AFB1 . This effect was not observed in yeast with intact cell walls, which may reveal a protective role of this structure against mycotoxins.

Effect of TNF\u03b1 stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Pharmaceutical biotechnological potential of filamentous fungi isolated from textile industry.

The need for more effective drugs for the treatment of infectious diseases as well as for general applications including wound healing and burn surgery, has guided efforts for the discovery of new compounds of medical interest. Microorganisms found in textile industrial waste have the ability to produce a variety of enzymes and/or secondary metabolites including molecules of pharmaceutical interest. The present work investigated the biotechnological potential of filamentous fungi isolated from textile industry wastewater for the production of collagenase and antimicrobial metabolites. From 28 isolates assayed, Sarocladium sp. ITF33 showed specific collagenolytic activity with values of 7.62 and 9.04Umg-1 for the intracellular and extracellular fractions, respectively. The isolate Penicillium sp. ITF28 showed the best antimicrobial activity, reaching MIC ranging from 1.0 to 0.0625mgmL-1 against five pathogenic bacteria. Molecular analyzes suggest that the isolate Sarocladium sp. ITF 33 can be considered a species not yet described. The results of the present work encourage studies of characterization and purification of the enzymes and secondary metabolites produced by the isolates found aiming future applications in the medical and pharmaceutical fields.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Neurite orientation dispersion and density imaging in evaluation of high-grade glioma-induced corticospinal tract injury

PurposeTo evaluate the application of neurite orientation dispersion and density imaging (NODDI) to brain glioma-induced corticospinal tract (CST) injury. Material and methodsTwenty-four patients with high-grade glioma (HGG) in or adjacent to the CST pathway and 12 matched healthy subjects underwent structural and diffusion MRI. The CSTs were reconstructed on the both sides. The CST features including morphological features (track number, average track length and track volume) and the diffusion parameter values including fractional anisotraphy (FA), mean diffusivity (MD), intracellular volume fraction (ICVF), isotropic or free water volume fraction (ISOVF) and orientation dispersion index (ODI) along the CST were calculated. The CST features were compared between the affected and healthy side for HGG patients and between the left and right side for healthy subjects. The relative CST features were compared across the healthy subjects, patients with motor weakness and patients with normal muscle strength. Receiver operating characteristic (ROC) curve was applied to evaluate the performance of each relative CST characteristic for HGG-induced CST changes. ResultsCompared with the CST features on the healthy side, the track number, track volume and FA along the CST changed significantly on the affected side for HGG patients (p < 0.05 for all), whereas MD and ICVF changed significantly on the affected side only for HGG patients with motor weakness (p = 0.012 for both). In patients with motor weakness, the relative MD was significantly higher (p < 0.001), whereas the relative FA and ICVF was significantly lower (p = 0.002 and <0.001) than those in patients with normal muscle strength. The relative ICVF had a similar area under curve (AUC) to that of MD (AUC=0.953 and 0.969). Compared with the relative CST features in the healthy subjects, only the relative ICVF was significantly lower in HGG patients with normal muscle strength (p = 0.012). ConclusionsNODDI seems to be useful in reflecting the HGG infiltration to CST, and can evaluate the CST destruction with a performance similar to DTI by providing additional information about neurite density for HGG-induced CST injury.

Dependence of Neuronal Network Structure on Event-Related Desynchronization Caused by Light Stimulation

Previous experiments have shown that neural population structure in the cerebral cortex affects cognitive function. However, it is unclear how the activity of the neural population is involved between them. In this study, we compared the relationship between the structure and activity of neural populations in the primary visual cortex. Intracellular volume fraction obtained by NODDI analysis of diffusion-weighted MRI was used as an indicator of neural population structure. The temporal delay in change of alpha wave intensity on event-related desynchronization in response to light stimuli was also used as an indicator of neural population activity. Using the stochastic oscillation mathematical model, we simulated event-related desynchronization and measured delay in change of alpha wave intensity when the coupling probability is changed on the model. The results showed that delay in attenuation of alpha wave intensity was smaller and delay in recovery was greater in both cases measurement and simulation, given higher neuron density. In conclusion, it is clear that structural features of the neuronal population affect the collective activity and that such phenomenon can be explained by mathematical models.

Anesthetic Preconditioning of the Small Intestine in Hemorrhagic Hypotension

Aim of the study. To investigate the preconditioning effect of sevoflurane on small intestinal mucosa in experimental hemorrhagic hypotension.Material and methods. The study was performed on a cohort of 106 male rats that included two experimental groups: one exposed to ether (Group 1, n=40) and another one exposed to sevoflurane (Group 2, n=40); two control groups included 20 intact animals, of which 10 were anesthetized with ether and 10 with sevoflurane. Six animals were excluded from the study because they died by the 2nd hour of hemorrhagic hypotension under ether anesthesia. The study parameters were measured at 15 min, 30 min, 1 h, and 2 h of hemorrhagic hypotension. Amylolytic activity of the small intestine mucosa was determined by E. A. Zabelinsky, B. W. Smith and I. M. Roe technique modified by A. M. Ugolev. The data were statistically analyzed using the nonparametric Mann-Whitney method.Results. By 15 min of hemorrhagic hypotension, the activity of amylase fractions in all small intestine regions in Group 2 animals was significantly lower vs the Group 1 rats. By 30 min of hemorrhagic hypotension, the activity of the enzyme fractions in all small intestine regions in Group 2 animals remained significantly lower than in Group 1, by an average of 2 to 9 times (P=0.01; P&lt;0.001), and after 1 h of hemorrhagic hypotension, it was 2 and 4 times lower (P=0.02; P&lt;0.001). By the 2nd hour of hemorrhagic hypotension, the activity of nearly all duodenal amylase fractions in the Group 2 animals remained 3-4 times lower compared to Group 1. Meanwhile, a significantly higher activity of slowly desorbing and intracellular amylase fractions vs the control group was observed in jejunum and ileum.Conclusion. In hemorrhagic hypotension under sevoflurane anesthesia, a decrease of the pancreas excretory function, stabilization of the brush border of the mucosa of all small intestine regions, including enterocyte membranes, was found during the first hour of experiment. Two hours after the hemorrhage, the biochemical evidence of brush border damage in the jejunum and ileum was revealed.

Exercise-induced fluid shifts are distinct to exercise mode and intensity: a comparison of blood flow-restricted and free-flow resistance exercise.

MRI can provide fundamental tools in decoding physiological stressors stimulated by training paradigms. Acute physiological changes induced by three diverse exercise protocols known to elicit similar levels of muscle hypertrophy were evaluated using muscle functional magnetic resonance imaging (mfMRI). The study was a cross-over study with participants (n = 10) performing three acute unilateral knee extensor exercise protocols to failure and a work matched control exercise protocol. Participants were scanned after each exercise protocol; 70% 1 repetition maximum (RM) (FF70); 20% 1RM (FF20); 20% 1RM with blood flow restriction (BFR20); free-flow (FF) control work matched to BFR20 (FF20WM). Post exercise mfMRI scans were used to obtain interleaved measures of muscle R2 (indicator of edema), R2' (indicator of deoxyhemoglobin), muscle cross sectional area (CSA) blood flow, and diffusion. Both BFR20 and FF20 exercise resulted in a larger acute decrease in R2, decrease in R2', and expansion of the extracellular compartment with slower rates of recovery. BFR20 caused greater acute increases in muscle CSA than FF20WM and FF70. Only BFR20 caused acute increases in intracellular volume. Postexercise muscle blood flow was higher after FF70 and FF20 exercise than BFR20. Acute changes in mean diffusivity were similar across all exercise protocols. This study was able to differentiate the acute physiological responses between anabolic exercise protocols. Low-load exercise protocols, known to have relatively higher energy contributions from glycolysis at task failure, elicited a higher mfMRI response. Noninvasive mfMRI represents a promising tool for decoding mechanisms of anabolic adaptation in muscle.NEW & NOTEWORTHY Using muscle functional MRI (mfMRI), this study was able to differentiate the acute physiological responses following three established hypertrophic resistance exercise strategies. Low-load exercise protocols performed to failure, with or without blood flow restriction, resulted in larger changes in R2 (i.e. greater T2-shifts) with a slow rate of return to baseline indicative of myocellular fluid shifts. These data were cross evaluated with interleaved measures of macrovascular blood flow, water diffusion, muscle cross sectional area (i.e. acute macroscopic muscle swelling), and intracellular water fraction measured using MRI.

Neurite orientation dispersion and density imaging parameters may help for the evaluation of epileptogenic tubers in tuberous sclerosis complex patients.

To investigate the usefulness of neurite orientation dispersion and density imaging (NODDI) in evaluating cortical tubers, especially epileptogenic tubers in tuberous sclerosis complex (TSC) patients. High-resolution conventional MRI and multi-shell diffusion-weighted imaging were performed in 27 TSC patients. Diffusion images were fitted to NODDI and DTI models. Tubers were visually assessed on different image types and scored by two neuroradiologists. For 10 patients who underwent epilepsy surgery, the contrast ratios between lesion and background tissue were measured on different image types, and these were compared between 16 epileptogenic tubers and 92 non-epileptogenic tubers. There were significant differences in lesion conspicuity scores and lesion-background contrast ratios across different sequences (both p< 0.001). The post hoc analysis showed that both the conspicuity scores and contrast ratios of intracellular volume fraction (ICVF) derived from NODDI were higher than other image types. For the 16 epileptogenic tubers, lesion visibility on ICVF was better/equal in 4/12 tubers compared with conventional MRI and better/equal in 5/11 tubers compared with DTI. Significant differences were observed between epileptogenic and non-epileptogenic tubers on diffusion maps, especially on orientation dispersion index derived from NODDI (p< 0.0001). ICVF demonstrated higher contrast than conventional MRI and DTI, which helped detection of subtle epileptogenic tubers. Moreover, NODDI parameters showed the potential to identify epileptogenicity. • The noninvasive localization of epileptogenic cortical tubers is essential for the preparation of epilepsy surgery for TSC patients. • ICVF derived from NODDI showed greater contrast than conventional MRI and DTI in detecting tubers, especially subtle epileptogenic ones. • Diffusion parameters, especially ODI derived from NODDI, can support the identification of epileptogenicity.

Albutate-1, a Novel Long-Circulating Radiotracer Targeting the Somatostatin Receptor Subtype 2

Currently, radiolabeled DOTA-[Tyr3]-octreotate (DOTA-TATE) is most commonly used in the clinic to image and treat neuroendocrine tumors. To further improve tumor uptake, and thus treatment, the amount of radiotracer that can accumulate in the tumor might be increased by prolonging the blood circulation time of the radiotracer. To achieve this, we designed Albutate-1, with both DOTA and an albumin-binding domain coupled to TATE via a suitable linker. The aim of this study was to determine the characteristics of the novel radiotracer Albutate-1. A competition binding assay was performed using [111In]In-DOTA-TATE on fresh-frozen SSTR2+ tumor sections. In vitro cell-uptake and internalization of [111In]In-Albutate-1 and [111In]In-DOTA-TATE were determined. The stability of [177Lu]Lu-Albutate-1 was tested. A biodistribution study was performed to provide tumor and organ uptake of [177Lu]Lu-Albutate-1. The biodistribution data was used to determine time-activity curves and the radiation dose for each organ and the tumor. The in vitro IC50 value of Albutate-1 was 1.2 nM. A higher amount of the tracer was found in the intracellular fraction than in the membrane fraction ([111In]In-Albutate-1 14.0 vs 1.9% of the added amount; [111In]In-DOTA-TATE 11.0 vs 1.5% of the added amount). After radiolabeling [111In] In-Albutate-1 was stable up to 3 days (93.1-88.9%) in labeling solution and very stable in mouse serum (90-94%) for at least 24 h. In vivo, [177Lu]Lu-Albutate-1 was cleared slowly from the circulation (1 h p.i. 58%ID/g, 168h p.i. 2%ID/g). The addition of an albumin-binding domain to DOTA-TATE extended the blood circulation to T1/2= 27.5h. The tumor absorbed dose was raised to 1455 mGy/MBq. Bone marrow, the dose-limiting organ in the mouse spine, unfortunately, received 765 mGy/MBq. All other organs also received a high radiation dose, reducing the therapeutic index.

Simultaneous multi-slice image reconstruction using regularized image domain split slice-GRAPPA for diffusion MRI.

The main goal of this work is to improve the quality of simultaneous multi-slice (SMS) reconstruction for diffusion MRI. We accomplish this by developing an image domain method that reaps the benefits of both SENSE and GRAPPA-type approaches and enables image regularization in an optimization framework. We propose a new approach termed regularized image domain split slice-GRAPPA (RI-SSG), which establishes an optimization framework for SMS reconstruction. Within this framework, we use a robust forward model to take advantage of both the SENSE model with explicit sensitivity estimations and the SSG model with implicit kernel relationship among coil images. The proposed approach also allows combining of coil images to increase the SNR and enables image domain regularization on estimated coil-combined single slices. We compare the performance of RI-SSG with that of SENSE and SSG using in-vivo diffusion EPI datasets with simulated and actual SMS acquisitions collected on a 3T MR scanner. Reconstructed diffusion-weighted images (DWIs) and the resulting diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) maps are analyzed to evaluate the quantitative and qualitative performance of the three methods. The DWIs reconstructed by RI-SSG are closer to the single-band ground truth images than SENSE and SSG. Specifically, the proposed RI-SSG reduces the normalized root-mean-square-error (nRMSE) against ground truth images by ∼5% and increases the structural similarity index (SSIM) by ∼4% compared to SSG. All three methods produce similar fractional anisotropy (FA) maps using DTI representation, but mean diffusivity (MD) and fiber orientation estimates using RI-SSG are closer to the reference than SENSE and SSG. RI-SSG results in NODDI maps with noticeably smaller errors than those of SENSE and SSG and improves the accuracy of the mean value of orientation dispersion index (ODI) by ∼5% and the mean value of intracellular volume fraction by ∼7% in regions of interest in brain white matter compared to SSG.

Inducing red pigment and inhibiting citrinin production by adding lanthanum(III) ion in Monascus purpureus fermentation.

Monascus pigments (MPs) are widely used natural colorants in Asian countries. The problems of low extracellular red pigment (ERP) and high citrinin remain to be solved in Monascus pigment production. The effect of lanthanum(III) ion (LaCl3) on Monascus purpureus fermentation was investigated in this study. The yields of ERP and biomass respectively reached maxima of 124.10 U/mL and 33.10 g/L by adding 0.4 g/L La3+ on the second day in the total 8-day fermentation; simultaneously, citrinin was decreased by 59.93% and 38.14% in the extracellular and intracellular fractions, respectively. Reactive oxygen species (ROS) levels were obviously improved by La3+ treatment, while the activities of catalase (CAT) and superoxide dismutase (SOD) were increased compared with the control. The ratio of unsaturated/saturated fatty acids in mycelia was increased from 2.94 to 3.49, indicating that the permeability and fluidity of the cell membrane were enhanced under La3+ treatment. Gene expression analysis showed that the relative expression levels of Monascus pigment synthesis genes (pksPT, mppB, mppD, MpFasB2, and MpPKS5) were significantly upregulated by La3+ treatment, and in contrast, the relative expression levels of citrinin synthesis genes (ctnA, pksCT and mppC) were markedly downregulated. This work confirmed that LaCl3 possesses the potential to induce red pigment biosynthesis and inhibit citrinin production in M. purpureus fermentation. KEY POINTS: • La3+ induced red pigment and inhibited citrinin production in Monascus fermentation. • La3+ regulated genes expression up for Monascus pigment and down for citrinin. • La3+ increased the UFAs in cell membrane to enhance the permeability and fluidity.

Evaluation of early microstructural changes in the R6/1 mouse model of Huntington's disease by ultra-high field diffusion MR imaging

Diffusion MRI (dMRI) has been able to detect early structural changes related to neurological symptoms present in Huntington's disease (HD). However, there is still a knowledge gap to interpret the biological significance at early neuropathological stages. The purpose of this study is two-fold: (i) establish if the combination of Ultra-High Field Diffusion MRI (UHFD-MRI) techniques can add a more comprehensive analysis of the early microstructural changes observed in HD, and (ii) evaluate if early changes in dMRI microstructural parameters can be linked to cellular biomarkers of neuroinflammation. Ultra-high field magnet (16.7T), diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI) techniques were applied to fixed ex-vivo brains of a preclinical model of HD (R6/1 mice). Fractional anisotropy (FA) was decreased in deep and superficial grey matter (GM) as well as white matter (WM) brain regions with well-known early HD microstructure and connectivity pathology. NODDI parameters associated with the intracellular and extracellular compartment, such as intracellular ventricular fraction (ICVF), orientation dispersion index (ODI), and isotropic volume fractions (IsoVF) were altered in R6/1 mice GM. Further, histological studies in these areas showed that glia cell markers associated with neuroinflammation (GFAP & Iba1) were consistent with the dMRI findings. dMRI can be used to extract non-invasive information of neuropathological events present in the early stages of HD. The combination of multiple imaging techniques represents a better approach to understand the neuropathological process allowing the early diagnosis and neuromonitoring of patients affected by HD.

Neurite Orientation Dispersion and Density Imaging of Rat Brain Microstructural Changes due to Middle Cerebral Artery Occlusion at a 3T MRI.

The purpose of this work was to demonstrate the feasibility of neurite orientation dispersion and density imaging (NODDI) in characterizing the brain tissue microstructural changes of middle cerebral artery occlusion (MCAO) in rats at 3T MRI, and to validate NODDI metrics with histology. A multi-shell diffusion MRI protocol was performed on 11 MCAO rats and 10 control rats at different post-operation time points of 0.5, 2, 6, 12, 24 and 72 h. NODDI orientation dispersion index (ODI) and intracellular volume fraction (Vic) metrics were compared between MCAO group and control group. The evolution of NODDI metrics was characterized and validated by histology. Infarction was consistent with significantly increased ODI and Vic in comparison to control tissues at all time points (P<0.001). Lesion ODI increased gradually from 0.5 to 72 h, while its Vic showed a more complicated and fluctuated evolution. ODI and Vic were significantly different between hyperacute and acute stroke periods (P<0.001). The NODDI metrics were found to be consistent with the histological findings. In conclusion, NODDI can reflect microstructural changes of brain tissues in MCAO rats at 3T MRI and the metrics are consistent with histology. This study helps to prepare NODDI for the diagnosis and management of ischemic stroke in translational research and clinical practice.

Intracellular and Extracellular Antibiotic Resistance Genes in Airborne PM2.5 for Respiratory Exposure in Urban Areas

The current research paradigm for the environmental dissemination of antibiotic resistance genes (ARGs) focuses on those harbored within bacteria (i.e., intracellular fraction). Understanding both intracellular and extracellular ARGs has particular implications for the flux and fate of ARGs that are airborne via fine particulate matter (PM₂.₅) from the ambient atmosphere to the human airway. In this study, we developed an operationally defined protocol for urban PM₂.₅ to quantify the abundance of ARGs occurring in free and phage-associated DNA, in addition to their counterparts commonly analyzed within bacterial cells. The results demonstrated that the extracellular fraction comprises a significant proportion of total ARGs in PM₂.₅, with subtype-specific dominance in either free or phage-associated DNA. A comparison between temperate and subtropical cities revealed geographical disparities of PM₂.₅-associated intracellular and extracellular ARGs due to the influences of regional meteorological factors and oxidative gases. Considering the potential for horizontal gene transfers and the efficiency of respiratory deposition, the extracellular fraction could represent >60% of the modeled inhalational intake of most of the analyzed ARGs in some cities. This study highlights the importance of the dynamics of ARGs in airborne PM₂.₅ and their health implications across climate zones and pollution gradients.

Toxic cyanobacteria in water supply systems: data analysis to map global challenges and demonstrate the benefits of multi-barrier treatment approaches

Abstract The occurrence of toxic cyanobacteria in surface waters and their impact on drinking water treatment plants (WTPs) is a growing, global concern. The main objective of this paper was to assess the presence of cyanobacteria in surface water sources and associated cell removal efficiency in full-scale WTPs across the world. Previously unpublished data was collected from WTPs experiencing cyanobacterial blooms in either their managed surface waters or recreational waters. In total, data were collected from 31 surface water sources and 21 WTPs in North and South America, Europe, Asia, and Australia. The most commonly detected species were identified in both the surface waters, including Microcystis, Anabaena, Nostoc, Oscillatoria, and Planktolyngbya, and water treatment plant intakes, including Microcystis, Cylindrospermopsis, Anabaena, Pseudanabaena, and Aphanizomenon. In the intakes, cyanotoxins and taste and odor (T&amp;O) compounds frequently co-occurred (80%) as did multiple cyanotoxins (39%). Conventional treatment saw a wide range of removal depending on the density of cells, species, and metabolites. Although more than 28% of sampling events displayed negligible or even negative removals of metabolites or cells due to accumulation within the clarifier, filtration, or water recycling, the presence of multiple treatment barriers, particularly advanced treatments like granular activated carbon and nanofiltration, allowed for the cells and their metabolites of concern to be removed to below guideline values. During treatment, total microcystins were often removed without releasing their intracellular fraction, whereas cylindrospermopsin, geosmin, and 2-MIB were commonly detected as entirely extracellular at the plant's intake. The maximum tolerable cell (MTC) counts for cyanotoxin- or T&amp;O-producing cells were calculated using guideline values, average removal efficiency, and the average cell quota derived from data. The 21 WTPs in this work were found to be able to tolerate approximately 74,000 cells/mL for microcystins, 8,000 cells/mL for cylindrospermopsin, and 1,200 cells/mL for geosmin and 2-MIB before exceeding guideline values. These levels provide guidance for water treatment plant operators to assess the potential risk associated with cells capable of producing cyanotoxins or T&amp;O compounds.

Different Antioxidative and Antiapoptotic Effects of Piceatannol and Resveratrol.

Resveratrol affords protection against reactive oxygen species (ROS)-related diseases via activation of SIRT1, an NAD+-dependent deacetylase. However, the low bioavailability of resveratrol limits its therapeutic applications. Since piceatannol is a hydroxyl analog of resveratrol with higher bioavailability, it could be an alternative to resveratrol. In this study, we compared the cytotoxicity, antioxidative activity, and mechanisms of cytoprotection of piceatannol with those of resveratrol. In C2C12 cells incubated with piceatannol, electrospray ionization mass spectrometry analysis showed that piceatannol was present in the intracellular fraction. A high concentration (50 μM) of piceatannol, but not resveratrol, induced mitochondrial depolarization and apoptosis. However, piceatannol at 10 μM inhibited the increase in mitochondrial ROS level induced by antimycin A, and this ROS reduction was greater than that by resveratrol. The reduction in hydrogen peroxide-induced ROS by piceatannol was also greater than that by resveratrol or vitamin C. Piceatannol reduced antimycin A-induced apoptosis more than did resveratrol. SIRT1 knockdown abolished the antiapoptotic activity of resveratrol, whereas it blocked only half of the antiapoptotic activity of piceatannol. Piceatannol, but not resveratrol, induced heme oxygenase-1 (HO1) expression, which was blocked by knockdown of the transcription factor NRF2, but not by SIRT1 knockdown. HO1 knockdown partially blocked the reduction of ROS by piceatannol. Furthermore, the antiapoptotic action of piceatannol was abolished by HO1 knockdown. Our results suggest that the therapeutic dose of piceatannol protects cells against mitochondrial ROS more than does resveratrol via SIRT1- and NRF2/HO1-dependent mechanisms. The activation of NRF2/HO1 could be an advantage of piceatannol compared with resveratrol for cytoprotection. SIGNIFICANCE STATEMENT: This study showed that piceatannol and resveratrol were different in cytotoxicity, oxidant-scavenging activities, and mechanisms of cytoprotection. Protection by piceatannol against apoptosis induced by reactive oxygen species was superior to that by resveratrol. In addition to the sirtuin 1-dependent pathway, piceatannol exerted nuclear factor erythroid 2-related factor 2/heme oxygenase-1-mediated antioxidative and antiapoptotic effects, which could be an advantage of piceatannol compared with resveratrol.

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses.

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.