Whether berberine mediates its anti-inflammatory and blood sugar and lipid-lowering effects solely by adjusting the structure of the gut microbiota or by first directly regulating the expression of host pro-inflammatory proteins and activation of macrophages and subsequently acting on gut microbiota, is currently unclear. To clarify the mechanism of berberine-mediated regulation of metabolism, we constructed an obese mouse model using SPF-grade C57BL/6J male mice and conducted a systematic study of liver tissue pathology, inflammatory factor expression, and gut microbiota structure. We screened the gut microbiota targets of berberine and showed that the molecular mechanism of berberine-mediated treatment of metabolic syndrome involves the regulation of gut microbiota structure and the expression of inflammatory factors. Our results revealed that a high-fat diet (HFD) significantly changed mice gut microbiota, thereby probably increasing the level of toxins in the intestine, and triggered the host inflammatory response. The HFD also reduced the proportion of short-chain fatty acid (SCFA)-producing genes, thereby hindering mucosal immunity and cell nutrition, and increased the host inflammatory response and liver fat metabolism disorders. Further, berberine could improve the chronic HFD-induced inflammatory metabolic syndrome to some extent and effectively improved the metabolism of high-fat foods in mice, which correlated with the gut microbiota composition. Taken together, our study may improve our understanding of host-microbe interactions during the treatment of metabolic diseases and provide useful insights into the action mechanism of berberine. With a growing increase in changes in diet, lifestyle and other factors, there has been an explosive epidemic trend of metabolic syndrome characterized by obesity, type II diabetes, non-alcoholic fatty liver disease (NAFLD), and atherosclerosis . As one Metabolic syndrome is typically accompanied by low-level, systemic chronic inflammation, leading to impaired insulin activity and the development of metabolic abnormalities . It has Bifidobacterium spp. that protect the intestinal barrier function and an increase in the abundance of pathogenic bacteria such as Desulfovibrio spp. that produce endotoxin and H2S, can compromise the barrier function of the intestine  as follows: Normal chow diet , NCD supplemented with berberine , high-fat diet , HFD supplemented with berberine , HFD supplemented with antibiotics (HFDABT group), and HFD supplemented with berberine and antibiotics (HFDBBRABT group). The mice were treated for 15 weeks, and drank freely during the experiment  were obtained from the Laboratory Animal Center of Shandong Academy of Medical Sciences  and fed on normal chow diet for 2 weeks to adapt to the environment. Thereafter, animals were divided into six groups , some liver tissues were fixed with 10% neutral buffered formalin for tissue section analysis and the rest were quickly frozen in liquid nitrogen and stored at −80°C until used for RT-PCR analysis. Five replicates of fecal samples were randomly collected from each group of mice and stored at −80°C for subsequent sequence analysis.Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed at the 16th week. Three mice in each group were randomly collected to OGTT and another three mice were used to ITT. Before the OGTT, mice were fasted for 8 h and then administered 2 g/kg body weight glucose orally. Blood glucose levels (BGL) were determined with a glucose meter  after 0, 15, 30, 60, 90, 120, and 150 min. Before the ITT, mice were fasted for 4 h and insulin (0.75 U/kg) was injected intraperitoneally. Blood glucose levels were determined, as described above, after 0, 15, 30, 60, 90, 120, and 150 min.Liver tissue was fixed in 10% neutral buffered formalin; then, it was processed routinely, embedded in paraffin wax, sectioned (4 μm thickness), and stained with hematoxylin and eosin (H&E). Histopathological assessment was conducted using a BX53 + DP26 light microscope  . Oil RedGAPDH). Primers for target genes, listed in ® Premix Ex Taq™ was purchased from Takara Biomedical Technology (Beijing) Co., Ltd. The amplification reactions were carried out on a LightCycler 480 Instrument (Roche Diagnostics GmbH) with an initial hold step (95°C for 10 min) and 45 cycles of a three-step PCR . After verifying that the amplification efficiencies of the selected genes and GAPDH were approximately equal, differences in expression levels were calculated using the 2–ΔΔCt method  according to the manufacturer’s protocol. The concentration of total RNA was determined using a NanoDrop One spectrophotometer . Then, 1 μg of total RNA was reverse transcribed into cDNA using oligo(dT) primers. Next, qRT-PCR was performed to analyze gene expression using the SYBR Green PCR Master Mix . The transcriptional levels of target genes were normalized against the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase . The V4–V5 hypervariable region of prokaryotic 16S rRNA gene was amplified using the universal primers 515F and 909R as previously described . The ampRaw reads were merged using the FLASH 1.2.8 software  and procpost hoc test was conducted using R 2.5.1  software  with Tukey–Kramer  R 2.5.1 . The Krusoftware . Non-parsoftware  was condsoftware  in R 3.5software . Kruskalsoftware . Principsoftware , and a hp < 0.05), whereas the body weight of HFDBBR mice was significantly lower than that of HFD mice in the end of the experiment  higher than those of the NCD mice, whereas the blood glucose levels of HFDBBR mice were lower than those of HFD mice , it still showed an increasing trend . However, the transcription levels of TNF-α in HFD mice were higher than those in NCD mice , indicating that berberine treatment biased macrophages toward M2 polarization , but was still higher than that of the NCD group. By comparison, the relative abundance of Bacteroidetes was significantly increased, although it was still lower than that in the NCD group . Berberine treatment did not significantly reduce the number of OTUs observed in the fecal microbiota of mice fed on a normal diet . However, berberine treatment significantly reduced the Shannon and Simpson indexes in these mice  and HFD significantly altered the composition of mice fecal microbiota . Among them, the fecal microbiota of mice fed on an HFD was significantly enriched for Mucispirillum, Odoribacter, Roseburia, Adlercreutzia, Oscillospira, Psychrobacter, Dehalobacterium, AF12, Coprococcus, Ruminococcus, and some unidentified genera . These bacteria did not show signs of significant enrichment in the mice fecal microbiota of the HFDBBR group. In contrast, Parabacteroides, Trabulsiella, Sutterella, Butyricimonas, Eubacterium, Coprobacillus, Robinsoniella, Bacteroides, Clostridium, Akkermansia, and Proteus were significantly enriched in the fecal microbiota of HFDBBR mice  among different groups. The relative abundances of genes in the butanoate, propanoate, and pyruvate metabolisms showed that the relative abundances of genes in the butanoate, and propanoate metabolisms were significantly reduced in the HFD group, and BBR treatment could alleviate the reduction, but it could not completely return to the control level . The relCox-2 is an inducible gene that is either not expressed or expressed in very low levels in most tissues or cells under normal conditions. However, extracellular stimuli, such as endotoxins and TNF-α, can induce the expression of COX-2 to help it exert its pathophysiological effects, which, in turn, can mediate an inflammatory cascade  and cells that are mainly involved in innate immunity  can participate in multiple physiological processes such as cellular communication, cell apoptosis, and the autoimmune response . When NO cascade . Reg3γ i cascade , we foun cascade . Liver m cascade . During  cascade . Macroph cascade . During  cascade . The che cascade . Moreove cascade . Here, wimmunity . These pimmunity . In thisMucispirillum and Psychrobacter  can be found below: The animal study was reviewed and approved by the Animal Ethics Committee of the Qilu Normal University.JJL, JLL, and HS designed the experiments. JJL, JLL, and SW performed the animal experiments. JJL and JN conducted gut microbiota analysis. JJL, CZ, JJ, GW, and HS measured biochemical data and conducted immunohistochemical analysis. JJL and HS wrote the draft of the manuscript. All authors revised and approved the final version of the manuscript.JN is an employee of Guangdong Meilikang Bio-Science Ltd., China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In their theragnosis algorithm, receptor somatostatin scintigraphy takes the central place, the diagnosis receptor somatostatin analogue (RSA) choice depending on laboratory experience and accessibility. However, in all cases, the results depend decisively on correct radiotracer tumoral uptake quantification, where unfortunately there are still unrevealed clues and lack of standardization. We propose an improved method to quantify the biodistribution of gamma-emitting RSA, using tissular corrected uptake indices. We conducted a bi-centric retrospective study on 101 patients with different types of NETs. Three uptake indices obtained after applying new corrections to areas of interest drawn for the tumour and for three reference organs  were statistically analysed. For the corrected pathological uptake indices, the results showed a significant decrease in the error of estimating the occurrence of errors and an increase in the diagnostic predictive power for NETs, especially in the case of lung-referring corrected index. In conclusion, these results support the importance of corrected uptake indices use in the analysis of  Neuroendocrine tumours (NETs) originate from the complex hormone-producing neuroendocrine system. This explains both common phenotype features and their great diversity in terms of key structural and functional phenotypic characteristics, molecular profile, localization, aggressiveness, type and site-specific prognosis and response to the treatment ,2. AlthoIn this context, functional NETs imaging emerged at an extremely fast and promising speed, a number of NETs phenotypic markers demonstrating their ability to be visualized with different PET or SPECT tracers . This cu1-SST5), with different tumoral proportions, depending on NETs type. For example, in lung NETs: SST1—63% to 83%, SST2—43% to 96%, SST3—5% to 54%, SST4—0% to 14%, SST5—0% to 71% [1—68%, SST2—86%, SST3—46%, SST4—93%, SST5—57% [2,5 have the highest affinity and are the mostly expressed in NETs, being the optimal target for the imaging diagnosis [Despite their heterogeneity, it is well known that 70% to 100% of NETs share the special mark of somatostatin receptor (SST) overexpression on the cell membrane surface ,9. Five SST5—57% . The SSTiagnosis . Therefoiagnosis ,14 and aiagnosis .111In -DTPA-octreotide, 68Ga-RSA, or 99mTc-RSA, such as 99mTc-EDDA/HYNIC-TOC (99mTc-TOC) [More than one RSA have been introduced along the time, both for SPECT and PET, as is the case of mTc-TOC) ,17. TheiThe accuracy of diagnostic methods is essential for the morphological and functional imaging evaluation in precision oncology. Subtle metabolic changes in functional imaging/pixel-based measurements may indicate a useful early response to therapy that have been found to precede any volume changes of the tumour . The useQuantitative functional imaging can contribute to understand of the mechanism of diseases, to assess the condition of the patient-based disease, in order to obtain the best therapeutic effect. Quantitative imaging parameters could be an important tool in personalised medicine, improving patient selection, identifying the population for which treatment would bring the greatest benefits, reducing unnecessary exposure and side effects .99mTc RSA uptake for the imaging personalised diagnosis of NETs. We propose a new background correction and quantitative functional uptake indices that can be useful both in diagnostic and theragnostic NETs approach.Given both the importance of accurate quantification of the tracer uptake and the paucity of systematic recent studies focusing on the field of NETs image quantification, this study aims to contribute to the improvement of the quantification method of the We conducted a retrospective multicentric study, which initially enrolled 107 patients previously diagnosed with NETs. The patients initially included were all patients sent for SRS investigation, over a period of 16 months (from December 2019 to March 2021), in one of the following different nuclear medicine units: Department of Nuclear Medicine, University Emergency Clinical Hospital “St. Spiridon” Iasi, Romania and Department of Nuclear Medicine, Clinical Centre of Kragujevac, Serbia.Prior to the research program, all approvals were obtained for conducting the study in compliance with the rules of ethics and deontology. When patients presented to the Departments of Nuclear Medicine for SRS, they all signed the informed consent, which includes a section on the use of data for research purposes.The names of the patients remained anonymous to the study centres.Patients aged between 18 and 80 years were previously diagnosed with NETs following structural imaging , neuroendocrine biomarkers assessment and biopsy examinations and were not treated with any SA over the investigation period, the time interval between SRS and the end of biological treatment being at least five weeks. Four patients with incomplete medical records were excluded from the study. Other two patients were excluded because they met certain exclusion criteria: the severe renal and hepatic failure, the inflammatory digestive diseases, . 99mTc-TOC SRS were enrolled in the study , fitted with a low-energy, all-purpose, parallel hole collimators and an energy window of 20%, set to 140 keV ± 15% were used. For all patients, early, 2, 4 and 24 h acquisitions were performed. Due to the better target-to-background ratio, the 4 h images have been chosen for quantification, being useful in differentiating normal bowel activity from pathologic lesions, with the muscle uptake and blood activity evidently reduced. Each patient received an activity of 10.57 MBq/bw of For whole body (WB) scans acquired at 4 h after tracer’s administration, a 256 × 1024 matrix was used, with zoom of 1 and 8 cm/min bed movement. For image processing/reconstruction parameters we used Syngo software, version Syngo MI Applications VA60C. Bk) . Linear, non-focal limited intestinal uptake was rated as nonspecific, nonpathological uptake. For the quantitative analysis of tumour and main reference organs  uptake, the data was analysed using a regions of interest (ROI) technique only on anterior views. Identical ROI of 245 pixels were quantified. For each pathological and physiological uptake areas considered to be of study interest and 1/3 upper right thigh was considered to be the reference background region (ROIBk) . For eacWe proposed a new background correction  which implies an activity correction related to the background, as shown in Bk from the tumour and the chosen reference organ ROIs and dividing the obtained value to the ROIBk value.In order to obtain corrected tumour (which represents in fact pure tumour uptake) or physiological uptake, our background corrections involved subtracting the ROIliver extraction from ROI TUliver was divided to ROIBk, necessary adjustment due to the overlap of intense hepatic uptake.For visualized tumour uptake at the liver level, the value obtained after ROIIt is important to use some universal values to quantify uptake (such as our indices), values that are independent of both the geometry of the acquisition and the individual differences between the patients.The possible variability given by these aspects was the main reason why we tried to find indices that actually analyse the relative quantitative value of pure tracer uptake at the tumour level, a capture that could be independent of the differences between patients.1 = ROItumour/ROIliver, I2 = ROItumour/ROIspleen, I3 = ROItumour/ROIlung), and their values were compared before and after the applied corrections, as shown in Three uptake indices were calculated (It test were applied. The Kolmogorov–Smirnov test (K-S) was applied to verify the normal distribution of the variables. For the series of normally distributed values, the pair t test was applied, and for the series that do not respect the normality condition the Wilcoxon matched pairs test was applied. The sets of compared values were pair values, coming from the same patients. However, the homoscedasticity of the series of compared values was tested. For this, the Levene test was applied. The results indicated that there were no significant differences between the variances (p > 0.05).Statistical data analysis was performed in STATA 16 software  and in SPSS 25 . Comparison tests applied to continuous numerical variables were selected based on the distribution of series values and the number of cases included in the analysis. For continuous numerical variables, the Wilcoxon matched pairs test and pair To compare the predictive value of uptake indices calculated according to the corrected vs. uncorrected formula, the receiver operating characteristic (ROC) curve and the AUC value (area under the ROC curve) were evaluated. This analysis was performed both according to the location of neuroendocrine tumours (NETs) but was also applied to the whole group.p) of the tests applied was considered to be 0.05. A p value lower than 0.05 indicated that there is a statistically significant difference.The reference value for the significance level (p = 0.4401) compared to the mean age of female patients (56.6 ± 11.2).The analysed group showed homogeneity in terms of distribution according to the gender of the patients . In addition, the mean age of male patients (54.8 ± 12.8) did not show significant differences . The corrected and uncorrected pathological uptake indices did not show a normal distribution (Kolmogorov–Smirnov test: p < 0.01). Thus, for comparison, the non-parametric test Wilcoxon matched pairs test was applied.We performed the quantitative analysis of the corrected and uncorrected pathological uptake indices , showed that the corrected values increased significantly compared to the values obtained based on the reference formula, without any applied correction . The corBk .1c, I2c and I3c uptake indices can be explained by the background correction  led to identifying a pure tumoral uptake ROI (ROIC TU). The significant increase of I1, the values obtained by correction are significantly higher (p < 0.05) for the primary MTC (p < 0.001), lung NETs (p = 0.0008) and pNETs (p = 0.0069). For the I2c uptake index, significantly higher values were highlighted for the lung NETs (p = 0.0002) and pNETs (p = 0.0161)  .3 uptake index. For all types of primary locations analysed, I3c values were significantly higher compared to uncorrected values .The predictive power was assessed considering the area under the curve (AUC) and the calculated estimation error. In the first part of the analysis, the tests were performed for the whole group (n = 101) , followe3c, the AUC value in this case increasing from 0.708 to 0.902 , the density in various proportions of SST99mTc RSA SRS in NETs diagnosis. It is evident that all these studies demonstrate the interest for this investigation in NETs diagnosis algorithm, and there is a great diversity on the radiotracer uptake quantification methods, and as a result, these cannot be correctly compared.A number of papers focused on the use of 99mTc RSA, the SST-overexpressing primary and metastatic NETs lesions can be detected, SRS imaging being more sensitive compared to structural methods  [There are data which reports that using ry NETs) .99mTc-HYNIC-TOC and 99mTc-HYNIC-TATE uptakes for 12 patients. They calculated target/background ratios using ROIs for the most active areas of tumour, liver, left kidney, with the right lung chosen as background. Their results showed that there was no significant difference regarding the tumour uptake, but only significantly higher liver uptake for 99mTc-HYNIC-TOC [3c, the reference organ for I3 being the lung. This difference could be explained by the fact that the summative uptakes were analysed by Cwikla et al., without a ROIBk subtraction correction.Cwikla and his collaborators compared YNIC-TOC . This is68Ga-DOTATOC, 99mTc-HYNIC-TOC and 111In-pentetreotide for SST NETs imaging. Their conclusion was that 99mTc-HYNIC-TOC is useful for SST characterization, similar to a good alternative to 68Ga-DOTATOC, having a higher imaging quality  and a lower radiation exposure for patients, compared to 111In-pentetreotide [Briganti, in 2019, published a comparative analysis between treotide .111In-DOTATOC (n = 6), 111In-DTPA octreotide (n = 4) and 99mTc EDDA/HYNIC-TOC (n = 10). Tumour/organ ratios were calculated, with ROI plotted for tumour, kidney, liver, spleen, heart and right thigh, on WB scans. The results obtained for 99mTc RSA were superior to 111In RSA, with the highest difference for the tumour/kidney and tumour/heart ratios , respectively the lowest for the tumour/liver ratio [In his semi-quantitative analysis on 10 patients, Decristoforo et al. compared er ratio . Unlike 99mTc-EDDA/HYNIC-TOC and 111In-DTPA-octreotide. ROIs for tumour, kidney, liver, spleen, heart and right thigh were plotted. Statistically significant differences (p < 0.001) were obtained for tumour/blood, tumour/liver and tumour/kidney ratios for 99mTc-EDDA/HYNIC-TOC compared to 111In-DTPA-octreotide uptakes [Bk was used.Gabriel and his collaborators performed a semi-quantitative analysis on 41 patients, using  uptakes . The stu99mTc-EDDA/HYNIC octreotate, with ROI plotted for each primary and metastatic NETs lesion, important organs  and adjacent normal tissue as background. This paper did not define only one reference ROIBk, the authors analysing the ratios between the target and the adjacent normal tissue. The study showed a high target/non-target ratio, with different values, related to the localization [Hubalewska-Dydejczyk analysed, also semi-quantitatively, 75 patients, using lization .99mTc-EDDA/HYNIC Octreotate with 111In-pentetreotide in 14 patients, Deveci et al. obtained, in their semi-quantitative analysis, significantly higher tumour/liver and tumour/kidney ratios and insignificant tumour/spleen and tumour/thigh ratios, showing that 99mTc-EDDA/HYNIC Octreotate is a good imaging method for NETs [Comparing for NETs .99mTc-TOC SRS images acquired at 4 h, for all patients, was related to the fact that pathological uptake at this time point was reported to show SST density more precisely than late uptake [In our study of 101 patients with NETs, the choice of quantification on e uptake . The hete uptake .We proposed the implementation of three uptake indices with correction, in order to improve the accuracy of SRS images interpretation by considering possible error sources. Statistically and comparatively analyse of the diagnostic predictive power of these quantitative uptake indices (corrected vs. uncorrected) sustained the utility of the method. Due to the fact that planar images evaluate 2D projections of a 3D scattering activity, the thickness and size of the source can raise issues . Pixel vBackground region localization choice is also important. Different backgrounds were used in different studies, such as liver, lung, bone, heart, thighs and mediastinum, sometimes related to the tumour localisation ,33. In o3c, the correction is specific, especially for pNETs, gastrointestinal and adrenal NETs, an index which, uncorrected, has no precision power, as can be seen from the data contained in p < 0.001). This is important in the case of small lesions Krenning 1 NETs, that, usually, do not receive PRRT, because it is considered that tumoral uptake lower than a hepatic one is correlated with a low SST expression. We observed that using background correction it was obtained a better target to non-target ratio and better contrast of images. This possibly means that this kind of NETs patients could be a potential PRRT candidate.Thus, our quantitative analysis showed that the applied correction brings great benefits for all three uptake indices, regardless of the type of NETs. The obtained results show that for IAn interesting situation is represented by Krenning 1 MTC lesions, where the uptake is low and the corrected uptake indices show an increased precision diagnosis power.2 uptake index increases its prediction , which suggests visible signs of predictive value improvement. The diagnostic estimation error decreased significantly in the case of I3c and, implicitly, significantly increased the diagnostic accuracy . A higher predictive value is noted when I3c was compared to I1c and I2c.Regarding the pheochromocytoma, the results obtained have no predictive power, but this aspect may be related to the small number of cases included, which indicates the need for further research on large samples to clarify this issue. However, it must be emphasized that the I2c, the diagnostic estimation error decreases compared to uncorrected I2 value. The analysis made for the correction to I3 shows a significant increase in the predictive value . The I1c and I2c values promise an increase in predictability and estimation accuracy, which would be more evident in an analysis performed on a larger sample of cases with pNETs. However important, I3c in this context of a small sample size, has a good accuracy. High accuracy and low diagnostic estimation error (p < 0.001) support our background corrections. In addition, in the case of gastrointestinal NETs, the quantitative analysis shows an I3c with a high accuracy and a low diagnostic estimation error (p < 0.001).For pNETs IRegarding the dimension of the chosen ROI, we have started from the known fact that the tumour environment is usually cellularly heterogeneous in relation with the malignant cellular markers’ distribution, including SST. As a consequence, the uptake of the radiotracer will be also heterogeneous in the whole tumour. If a ROI that covers the entire tumour will be drawn, cells with a lower number of SST receptors and those with a higher number of receptors will be both included; in this situation, the degree of global radiotracer uptake will quantitatively reflect the totality of the receptors in that tumour. Subsequently, this will be related with the therapeutic tracer uptake in the following PRRT, which will affect all the SST from the tumour by beta radiation. If a small tumoral ROI, which will include only the most intense uptake area of the tumour, will be drawn, the rest of the cells that still express receptors, even if in a smaller number, will be lost from the quantification. This can affect the PRRT monitoring efficiency. This was the reason why the chosen size of ROI was a compromise which covered mostly of tumour sizes of studied patients.Our analysis shows that the use of uncorrected ROIs is followed by a high probability of errors, their cause being these summative uptakes themselves. The error will impact the diagnostic process of this pathology already known to be extremely heterogeneous, greatly delaying the correct diagnosis. This situation can lead to a late or erroneous use of various therapies, not necessarily in the correct version, with multiple disadvantages for the patient both in the short and long term. 18FDG-PET [Image processing with quantitative evaluation is standardized, until now, more for PET imaging, involving the use of standardized uptake value (SUV), metabolic active tumour volume and glycolysis of tumour lesion ,35. If s8FDG-PET  is usefuThe major limitation of the present study, similar to most published works, is the lack of the histological validation for all patients included. Our results may be of great help in terms of methods, representing the first step for further validation studies. The results are promising for an accurate quantitative assessment of the pathological uptake of gamma-emitting tracers to improve the diagnostic accuracy. They can have great applicability in one of the most important directions of development of imaging methods, artificial intelligence, where quantitative imaging data could successfully support “virtual biopsy” , bringin99mTc RSA remain a valuable choice in terms of cost, effectiveness and accuracy of NETs diagnosis. The use of the corrected uptake indices can significantly improve the accuracy of SRS NETs diagnosis, decreasing the variability and possibility of errors, making possible a careful implementation of a precisely targeted personalised therapeutic approach of the patients.


We have prepared four different samples, each one annealed at different temperatures, in order to produce different mean NP sizes, ranging from 26 nm up to 106 nm. Magnetization measurements revealed a FC-ZFC irreversibility and from the coercive field as function of temperature we determined the blocking temperature. A phase diagram was delivered as a function of the NP mean size and, based on this, the heating mechanism understood. Small NPs (26 nm) is heated up within the paramagnetic range of temperature  mean size, for the manganite composition La Further studies in animals have shown, as a result of heating by magnetic nanoparticles (NPs) in an alternating magnetic field, the reduction in size and disappearance of tumors [Local magnetic hyperthermia is one of the promising methods for cancer treatment ,2,3,4,5.f tumors ,10.0.75Sr0.25MnO3 (LSMO) manganite for the present study, since the critical temperature of the bulk material is close to 330 K [The ability to control the magnetic properties of NPs is of special importance. Namely, the critical temperature of NPs, in practice, avoids overheating of the magnetic fluid. This nuance is strategic from medical point of view, since overheating of a few degrees can damage living tissues. Blocking temperature (for superparamagnetic particles) depends on relaxation time and determines the conditions in which thermal fluctuation energy is comparable with magnetic anisotropy energy. It is shown in reference  that heato 330 K .In this work, our goal is to understand how the NPs size interferes on the heating production, for hyperthermia applications. Thus, samples of LSMO with different NP were produced, in order to change the heating parameters . Thus, tSample synthesis: La0.75Sr0.25MnO3 (LSMO) samples were synthesized by sol-gel method (Pechini) [Pechini) ,14, usinPechini) . For theX-ray diffraction: The structural characterizations were performed through X-ray diffraction using a Brucker D8 Advances, Brucker Corporation, Billerica, MA, USA  JEOL 2100F, JEOL Ltd., Tokyo, Japan (200 kV) at Brazilian Center for Research on Physics—CBPF. We have used the software ImageJ to obtain the NP distribution.Magnetization: Magnetization was measured using a SQUID magnetometer from Quantum Design at Laboratory of Materials and Low Temperatures, State University of Campinas and Faculty of Physics, Lomonosov Moscow State University—MSU.Heating-cooling curves: Heating power measurements were carried out at Faculty of Physics-MSU on the reconfigurable setup for high frequency magnetothermal measurements , with the starting lattice parameters for the bulk Normalized X-ray diffraction (XRD) patterns for all samples prepared and the refinement results are presented in ructures . The Rie3.3743 Å . From thk is a constant close to 1, U, V and W are related to the full width at half maximum (FWHM) of the Pseudo-Voight function used to fit the XRD patterns [From the XRD results, it is possible to estimate the mean crystallite size patterns . The valequation:DS=kλβcosIn order to confirm the structural analysis, Transmission Electronic Microscopy (TEM) images were acquired for all samples see . Figure D represents the NP size, A stands for the amplitude; while the parameters Above,  NPs, as :(3)〈D〉=eUsing these relations, the mean particle size was calculated for all samples prepared and are presented in a-axis. As given in a-axis. For the particular case of the 106 nm sample, the high resolution images give a clear visualization that the super-lattice is close to the observed for the bulk counterpart [Furthermore, a magnification reveals the fine crystalline feature of the structures and allows the measurement of the d-spacing, depicted on the inset of nterpart . In addiField Cooled (FC) and Zero Field Cooled (ZFC) curves for the samples under consideration are shown in superparamagnetic region);paramagnetic region).Note the room temperature is an important reference temperature, because the heating experiments start from this base point. Further discussion about this topic will be delivered further in this article.c is the specific heat of the distilled water, m and M represent, respectively, the fluid and samples masses. In addition, From these heating-cooling curves for all samples prepared, the Specific Absorption Rate-SAR could be obtained through the following relation:ng curve , obtaineng curve , where tf is the frequency of the radiation applied over the system, Above, The frequency dependence for SAR in sensweig , consideSmaller NPs, with mean diameter size of 26 nm, were heated up within the paramagnetic region and therefore the relaxation time is expected to be much faster than the reference case above described. Thus, for this limiting case, we expect Equation  tends toBigger NP of mean diameter size of 58 nm and 106 nm are heated up within the magnetically blocked region (T<TB see b. This a3O4 (magnetite) is among the systems with the greatest potential for several biomedical applications [2O3 are also used for magnetic hyperthermia application due to its low toxicity, high saturation magnetization and tuned Curie temperature [2O3 suppresses the transition to the antiferromagnetic 2O3, but there is a reduction on the magnetization. The antiferromagnetic suppression transition revealed to be efficient in the destruction of cells, with the disruption of the cytoskeleton [5Si4 in its way has a 0.75Sr0.25MnO3 nanoparticles, they present low toxicity, biocompatibility, and a high SAR value; however, they also tend to aggregate [Furthermore, to compare with the reported values in literature, ications ,33. The ications . Similarperature . The preskeleton . Regardiegulated . Howeveregulated ,36. As fggregate . By incr0.75Sr0.25MnO3 by handling the annealing temperature of the samples. We obtained four samples, with mean NP diameter size ranging from 37 nm up to 106 nm—values obtained from Transmission Electronic Microscopy (TEM), with fine crystalline features.We have prepared nanoparticles (NPs) of lanthanum strontium manganites with the composition LaMagnetization measurements reveal a remarkable ZFC-FC irreversibility; and an analysis of the coercive field as a function of temperature , for all samples produced, allows the determination of the blocking temperature. From these characteristic temperatures, we made a phase diagram for this system, as a function of mean NP diameter size. We observed that the smaller NP (26 nm) is heated up within the paramagnetic range of temperature (


Pseudomonas species have shown a low-resolution rate, without a high resistance rate to antipseudomonal antibiotics. This suggests that bacterial virulence factors can act and influence peritonitis evolution. This study aimed to describe the microbiological characteristics of NF-GNB causing PD-related peritonitis and analyze their influence on the outcome. (2) Methods: We analyze the 48 isolates from NF-GNB peritonitis, which were stored in our culture collection regarding bacterial resistance, biofilm, and other virulence factors’ production, and clonal profile. Additionally, we collected data on treatment and outcomes from patients’ clinical registers. (3) Results: The etiologies were species of Pseudomonas (50%), Acinetobacter (36%), and other NF-GNB (14%). There was a high (75%) proportion of biofilm producer lineages. The in vitro susceptibility rate of Pseudomonas spp. to amikacin, ciprofloxacin, and ceftazidime was significantly greater than that of Acinetobacter spp. and other species; however, there was a similar low-resolution rate (<45%) among the episodes attributable to them. Pseudomonas species have a polyclonal profile, while we found a clone of five multiresistant Acinetobacter baumannii over an 8-year interval (2000–2008), which suggest an origin from the healthcare environment. (4) Conclusions: We are not able to identify any predictor of outcome, but it is possible that biofilm and others virulence factors can act in concert and contribute to the bad outcome.(1) Background: Peritonitis due to nonfermenting Gram-negative bacilli (NF-GNB) is a dramatic complication of peritoneal dialysis (PD) with bad outcomes. Previous studies of PD-related peritonitis due to  Despite the substantial decrease in peritonitis rates over recent decades ,2, it reStaphylococcus aureus [Usually, PD-related peritonitis progresses to resolution ; howevers aureus  and Grams aureus ,10.Pseudomonas species showed poor outcomes. The first included 191 Australian patients and reported high rates of catheter removal (44%) and hemodialysis transfer (35%) [The reasons for the unfavorable outcome of NF-GNB peritonitis are not fully known. Despite the indisputable role of bacterial resistance, this does not seem to be the only property influencing the outcome. The two largest previous series on PD-related peritonitis caused by er (35%) . The secer (35%) .Pseudomonas species, that can produce exoenzyme S, an inhibitor of protein synthesis; hemolytic phospholipase C, which is related to the destruction of cell membranes; exotoxin A, a promoter of tissue destruction and macrophage response inhibition; alkaline protease, a causer of tissue damage and inactivation of IgG; elastase, an immunoglobulin degradation factor; rhamnolipids, which are associated with bacterial adhesion; and alginate, which actively participates in the production of the biofilm, a polysaccharide matrix that surrounds the microorganisms, forming colonies; along with other virulence factors. Biofilm formation represents a protective growth mode that allows microorganisms to survive in hostile environments. Clinically, biofilms are responsible for many persistent and chronic infections because of their inherent resistance to antimicrobial agents, due to the difficulty antimicrobials find in penetrating this protective matrix [NF-GNB produces several virulence factors, particularly e matrix ,16,17,18A previous study by our group was not able to identify which clinical factors, treatments employed, or microbiological factors of the NF-GNB causing peritonitis on peritoneal dialysis were related to the outcomes of this infection; only exit-site infection (ESI) was identified as an independent predictor of non-resolution of these infections . Then, tFrom 1997 to 2015, there were 70 episodes of NF-GNB peritonitis. We retrieved 48 isolates from these episodes from our culture collection for microbiological testing. The microorganisms isolated and patient information are listed in P. aeruginosa in the years 1998–1999 (seven cases) and in the year 2006 (seven cases). In relation to the 48 episodes, 50.0% (24 cases) were caused by Pseudomonas spp., 37.5% (18 cases) were caused by Acinetobacter spp., 6.2% (three cases) were caused by Achromobacter spp., 4.2% (two cases) were caused by Burkholderia spp., and one case (2.1%) by Stenotrophomonas maltophilia  [The epidemiology of these isolates in relation to the studied period was varied; we only observed a concentration of episodes caused by With respect to the features of the patients, 56.2% were male, and 56.2% were below 50 years of age . In the Pseudomonas species had a higher susceptibility rate than Acinetobacter species to all antimicrobials, except for imipenem. Additionally, Pseudomonas had a higher susceptibility rate than Achromobacter species to amikacin, ciprofloxacin, and cefepime. B. cepacia and S. maltophilia were tested only for ceftazidime and were susceptible  with resistance profiles to more than three classes of antibiotics, namely eight A. baumannii, two P. aeruginosa, one A. haemolyticus, and one B. gladioli. The isolates from the years 2013 to 2015 showed sensibility to all the antibiotics tested. Therefore, we did not observe significant changes in the resistance pattern of these isolates during the entire period studied.In the period studied, we observed that the isolates causing peritonitis in the years 1997 to 1998 were sensitive to all antibiotics tested in this study, with only one Pseudomonas isolates, 11 Acinetobacter isolates, 1 Achromobacter isolate, and 1 S. maltophilia isolate. Two Pseudomonas isolates, seven Acinetobacter isolates, two Achromobacter isolates, B.cepacia, and the B. gladioli isolate were not biofilm producers. Results reproduced from Dos Santos et al. [Of the 48 samples, 35 (75%) were biofilm producers, including 18 strong, 7 medium, and 10 weak biofilm producers. The biofilm producers consisted of 22 s et al. .P. aeruginosa were positive for the alginate D, hemolytic C, non-hemolytic phospholipase C, exotoxin A, alkaline protease, and elastase genes, in contrast with 21 (87.5%) for rhamnolipids and 14 (58.3%) for exoenzyme S.All isolates from P. aeruginosa and A. baumannii due to their higher prevalence. Only 17 P. aeruginosa isolates and 9 A. baumannii isolates were included, because at the time of the pulsed-field gel electrophoresis (PFGE) analysis, it was not possible to recover the other isolates.This analysis was performed for the species P. aeruginosa profile  treatment was based on ISPD guidelines for empirical  ,20,21, wmmended, ; for thePseudomonas spp., the adjusted treatment was: two antipseudomonal drugs in 23 cases (97.8%)  and no adjustment in one, in which immediate catheter removal occurred. We observed an episode caused by S. maltophilia, in which ceftazidime plus ciprofloxacin was used for adjustment. For the 18 Acinetobacter spp. Episodes, the adjustment occurred in 100.0% of the cases and was based on monotherapy with imipenem in nine cases, amikacin in eight cases, and ceftazidime in one case. The duration of antibiotic therapy was at least 21 days [In our sample, for 24 episodes caused by 3 cases 9.8% , caused by one isolate of A. baumannii and one isolate of A. ursingii.Of the 48 episodes, we found 13 resolutions (27.1%); 4 relapses (8.3%); 17 refractory peritonitis episodes (35.4%); 12 catheter removals on the fifth day of treatment (25.0%) that occurred in episodes caused by seven isolates of Pseudomonas, 38.8% for the 18 caused by Acinetobacter, and 16.6% in peritonitis caused by other NF-GNB species (p = 0.35).The resolution rate was 20.8% for the 24 infections caused by p = 0.11). Therefore, we are not able to apply a multiple regression model.Only biofilm production was associated with the dependent variable of non-resolution of peritonitis  to the majority of antibiotics, except for ciprofloxacin. These findings suggest that bad outcomes observed in this study (resolution rate < 40%) and in the two largest previous series [Pseudomonas species, are a consequence of its aggressive virulence profile beyond antibiotic resistance.Our results confirm a high production of biofilm among NF-GNB, in particular, s series ,12, whicHowever, biofilm production is a potential determinant of an unfavorable antimicrobial response. The routine antibiotics prescription is based on the MIC of the drug for planktonic cells, which are more sensitive to antimicrobials than their counterparts wrapped in a biofilm ,30. We fWe were not able to identify a single virulence factor as a predictor of the outcome, and this analysis was hampered by the finding of their production in almost all isolates. Although individual analysis of the pathogenic factors did not show an association with the outcome, this does not rule out the possibility that pathogenic action of these may contribute to a bad outcome.Acinetobacter, and their resistance to several antimicrobials was confirmed in this series, except for imipenem, and this becomes a major therapeutic challenge [A. baumannii with five samples, isolated within an interval of 8 years with a multi-resistance profile to antimicrobials. These bacteria are commonly found in hospital environment infections [Acinetobacter species cause PD-related peritonitis, which is difficult to treat, with a high rate of catheter removal and technique failure [P. aeruginosa species presented a polyclonal profile suggesting different origins, probably from the patient’s microbiota or their environment.There was an important proportion of episodes due to species of hallenge . Clonal fections , and the failure ,35. In cAchromobacter species, B. cepacia, and B. gladioli, which have been rarely described as PD-related peritonitis etiologies. The precise identification of NF-GNB represents a challenge for conventional microbiology, due to the phenotypic similarity and taxonomic complexity of these agents. Phenotypic tests based on morphology and biochemical characteristics often provide erroneous identifications. In our study, this was minimized with the identification of isolates by means of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), a technique that identifies microorganisms based on their protein profile.We identified microorganisms such as the P. aeruginosa), we refrozen these strains again. When we went to recover them again to perform the clonal typing of the isolates, for reasons unknown to us, seven strains of P. aeruginosa and two strains of A. baumannii did not grow again. Because we have had a few isolates included in the study, we left the results of the other techniques for all isolates (48 strains), without excluding the strains not included in the PFGE technique. However, to our knowledge, it was the first study that addressed the virulence factors intrinsic to these germs and their potential role in outcomes. In addition, it provided novel information about pathogens rarely identified as the genus Achromobacter, which suggests the benefits of the use of new techniques such as MALDI-TOF for bacteria identification in peritonitis.Our study has several limitations, in addition to the small sample size. The financial resources for this study came from a grant from the by São Paulo Research Foundation (FAPESP), which ended in 2015. As the microbiological assays that were performed are not used in daily routine practice, it was not possible to proceed with sample follow-up after 2015. During this study, we performed the laboratory techniques at different times, and the last performed was the clonal typing of the isolates (PFGE). Initially, we recovered the 48 isolates that were stored at −80 °C, and after performing the first techniques  [The stored samples were re-isolated on MacConkey agar plates (Oxoid) and reidentified. The isolates were Gram-stained to confirm purity and to determine morphology and specific color. Afterwards, they were identified by conventional biochemical methods . In addi France) .We tested the susceptibility to amikacin, ciprofloxacin, cefepime, imipenem, and ceftazidime by means of the minimal inhibitory concentration (MIC) obtained by the E-test , made on the basis of the Clinical Laboratory Standards Institute (CLSI) based on the 2019 Clinical Laboratory Standards Institute (CLSI) breakpoints .8 CFU/mL). In addition, we inoculated a well only with culture medium to be used as a reading standard (blank). The inoculated plates were incubated at 37 °C for 48 h and then washed with Phosphate-Buffered Saline 4 times to remove non-adherent bacteria. Adherents were fixed with formalin (2%), and after 20 min, they were removed, and the preparations were washed 4 times with water. Further to this, the preparations were stained with a crystal violet solution (1%) for 20 min, after which, they were washed 3 times with water to remove excess dye. After the drying period, the dye was solubilized with methanol for 10 min, and the optical density (OD) measured at 540 nm was determined [The samples were grown in TSB at 37 °C for 18 h. To assess the bacterial ability to adhere to abiotic surfaces, we used polystyrene plates with 96 wells, each of which added 200 µL of TSB and 10 µL of the bacterial suspension , elastase (lasB), hemolytic phospholipase C (plcH), non-hemolytic phospholipase C (plcN), exoenzyme S (exoS), exotoxin A (toxA), alkaline protease (lasB), and rhamnolipid (rhlAB). The protocol, for each reaction and primer, was performed as described by Lanotte et al. [We used the polymerase chain reaction (PCR), in which the samples were evaluated for the presence of the genes encoding alginate , respectively. We followed the protocol by Durmaz et al. [A. baumannii and the PulseNet protocol , serotypes of Salmonella, Shigella sonnei, and Shigella flexneri) for P. aeruginosa. The DNA profiles generated by PFGE were inserted into the BioNumerics software, version 7.6  for analysis. Dendrograms were built for each species, with optimization and tolerance parameters set at 1.0 and 1.5, respectively. Two or more isolates with a similarity coefficient of 80% or higher were chosen for the definition of clusters.The z et al.  for A. bAntibiotic treatment and microbiological characteristics were studied regarding their association with the peritonitis outcome. Using the International Society for Peritoneal Dialysis (ISPD) criteria , the outp value > 0.20 as the elimination criterion from univariate analysis. A p value < 0.05 was considered significant.We compared frequencies using the chi-square test or Fisher’s exact test and used binary logistic regression with backward stepwise to determine predictors of outcome, which were classified in two mutually results: resolution or nonresolution . To select the variables to multivariate model, we used a Pseudomonas species, is not enough to explain the bad outcome. No individual virulence factor was associated with the outcome, which does not rule out the possibility that they will act in concert, impairing the response to antimicrobial therapy. The high prevalence of multi-resistant Acinetobacter species causing PD-related peritonitis, and with a clonal profile suggesting hospital origin, raises alarm about the care for the prevention and management of these infections.Our results confirm that NF-GNB PD-related peritonitis is a serious infection with a very low-resolution rate. Bacterial resistance, particularly among episodes caused by


Age-related macular degeneration (AMD) is a multifactorial disease, which can culminate in irreversible vision loss and blindness in elderly. Nowadays, there is a big gap between dry AMD and wet AMD on treatment. Accounting for nearly 90% of AMD, dry AMD still lacks effective treatment. Numerous genetic and molecular researches have confirmed the significant role of the complement system in the pathogenesis of AMD, leading to a deeper exploration of complement inhibitors in the treatment of AMD. To date, at least 14 different complement inhibitors have been or are being explored in AMD in almost 40 clinical trials. While most complement inhibitors fail to treat AMD successfully, two of them are effective in inhibiting the rate of GA progression in phase II clinical trials, and both of them successfully entered phase III trials. Furthermore, recently emerging complement gene therapy and combination therapy also offer new opportunities to treat AMD in the future. In this review, we aim to introduce genetic and molecular associations between the complement system and AMD, provide the updated progress in complement inhibitors in AMD on clinical trials, and discuss the challenges and prospects of complement therapeutic strategies in AMD. Age-related macular degeneration (AMD) is a significant cause of irreversible blindness and vision impairment among the elderly in developed countries , 2. WithThe pathogenesis of AMD is complex. Although the identification of genetic risk factors for AMD has been quite successful, the mechanisms by which these risk factors interact in AMD are still unclear. Thus, the discovery of future therapeutic strategy for AMD is still full of challenges. Nowadays, there is still a big gap between dry AMD and wet AMD on treatment. On the one hand, wet AMD is currently treatable. Monthly intravitreal injections of anti-VEGF drugs  have become the first-line treatment for wet AMD, which can reduce the incidence of severe vision loss significantly . HoweverNumerous genetic studies and molecular research have confirmed the significant role of the complement system in AMD, including genetic variants, overactivation of alternative pathway, inflammation, oxidative stress, lipid accumulation, and energy metabolism. As the links between the complement system and AMD are becoming clearer, many innovative therapeutic attempts targeted at complement components emerge in the therapy of AMD. To date, there is still a lack of up-to-date and comprehensive papers on the involvement of the complement system in the pathogenesis of AMD and the research actuality of complement inhibitors in the treatment of dry AMD versus wet AMD, even though there have been a number of studies and review articles , 10. ThiThe complement system, first described as an auxiliary system by Jules Bordet and Paul Ehrlich in the late 19th century, is a highly regulated protein network which can be activated in a cascaded manner, and acts at the interface of innate and adaptive immunity . To some2O) in the fluid phase, also known as “tick-over,” and by C4b2a (C3 convertase) to cleave C3 into C3a and C3b in the solid phase [2O) and C3b can be bonded by factor B (FB) and factor D (FD) to form C3(H2O)Bb and C3bBb, respectively. Of note, AP has a positive feedback on the generation of ample amounts of C3b, which is regarded as the amplification loop between C3bBb and C3b for the other two pathways , lectin pathway (LP), and alternative pathway (AP)  Figure . Specifiid phase , 15. Subpathways . ConsequIn the final stage, the convergence of the C3 convertase and additional C3b results in the formation of membrane-bound enzyme complexes (C5 convertases), which integrate all processes into a common terminal pathway. The C5 convertase will cleave C5 into C5a and C5b afterwards. Furthermore, owning a new short-lived binding site, C5b sequentially recruits C6, C7, C8, and C9 to form the terminal complex C5b-9, also referred to as the membrane attack complex (MAC). Formation of MAC will lead to a pore in the target cell membrane and ultimately causes cell lysis and death, which can be downregulated by complement inhibitors like CD59 , 17.CD59 is a naturally existing inhibitor of MAC formation that functions by binding to the terminal complement protein complex, preventing C9 molecules from binding to the cell membrane, and thereby forming pores . The comAs an incredibly complex, multifactorial disease, AMD is driven by a combination of natural aging, unhealthy lifestyle, and genetic predisposition. With increasing age, mitochondria within the RPE cells of AMD patients decrease in size and number and produce more waste products , 23, whiThe relevance between AMD and complement system has been revealed in numerous researches since the 1980s, both locally and systemically: (a) the existence of complement components in drusen, for example, C3a and complement factor H (FH) , 38–42; As an important inhibitor of the complement system, complement FH can not only compete with FB to bind C3b, accelerating the dissociation of the alternative system C3 convertase (C3bBb) resultingly, but also act as a cofactor for facilitating the FI-mediated C3b inactivation , 57. GenAs mentioned above, C3 is the key component among all complement pathways. Any functional changes in C3 can directly influence the downriver cascade. Therefore, C3 gene is another important genetic variant in AMD. R102G variant is the most commonly happening mutation in the C3 gene of AMD patients . The polThe complement FI gene contains a serine protease domain which is the cause of cleaving and inactivating C3b and C4b to regulate complement activation. To date, there are 110 variants in the complement FI gene discovered in AMD patients, among which nine are nonsense changes, one is frameshift change, and 100 are missense changes in the FI protein . A case-Both serine proteases FB and FD have significant roles in the formation and activity of the C3 convertase in AP , 76. Of C9 is the most downstream component of the terminal complement pathway. A remarkable decreasing of C9 can affect the production of MAC and finally decrease the cytolytic activity –83. At pTaken together, on the one hand, these findings suggest that genetic variants that hinder the negative regulation of the complement system promote the development of AMD. Notably, it has been suggested that the additive effect of risk variants leads to an aggregated risk of disease . On the While genetic studies have been somewhat successful in determining the risk of the complement system for the development of AMD, the understanding of how these variants drive AMD at the molecular level remains largely incomplete. Research on the molecular mechanisms of the complement system will not only provide a deeper understanding of AMD but will also contribute to the discovery of future drug treatments. To date, molecular studies of the complement system in AMD have focused on following main areas: chronic inflammation, oxidative stress, lipid accumulation, and energy metabolism.The most notable consequence of complement activation is that it can mediate the recruitment and activation of immune cells, such as microglia, monocytes/macrophages, lymphocytes, and mast cells, through the release of complement components C3a and C5a , 93. In Drusen is a characteristic fundus change in AMD, and it is now known that lipids are a major component of drusen . Indeed,2O2, smoke extracts, and lipid oxidation products [In recent years, several studies have exposed RPE cells to different types of stress stimuli separately, measuring and analyzing the levels of complement proteins and complement factors. For example, extracellular levels and genetic levels of C3 were significantly improved when human adult retinal pigment epithelial cells (ARPE-19) were exposed to Hproducts –102. In products .Recently, imbalances in energy metabolism within RPE cells have been considered as an important aspect of AMD pathology. An experimental animal study showed abnormally large mitochondria, reduced levels of mitochondrial DNA, and decreased ATP production in photoreceptors and RPE cells in FH knockout mice , 105. ThThe current treatment strategy for AMD is not optimistic. Although anti-VEGF injection is an effective treatment option for wet AMD, there is still a proportion of patients who are not sensitive to the drug. In addition, even with monthly anti-VEGF therapy, GA still remains an inevitable long-term progressive outcome for a majority of patients with wet AMD , 108. HoIn the past decades, as the first FDA-approved complement inhibitor, eculizumab (C5 inhibitor) is used to treat the hemolytic disorder, paroxysmal nocturnal hemoglobinuria (PNH), which remarks an important milestone in complement drug discovery . Since tAlthough numerous studies have explored the etiology and pathogenesis of dry AMD, there is still a lack of an effective strategy for the treatment. Therefore, compared with wet AMD, it is more urgent to devise an efficiently and feasible treatment for dry AMD. According to the statistics, in dry AMD, there are at least 13 different complement inhibitors which have been applied in nearly 30 different clinical trials . Next, wNCT00473928). However, the trial was terminated prematurely due to the deposit formation of drugs in the eye of GA in phase II trial (NCT01603043). The second is APL-2, also known as pegcetacoplan, another peptide compstatin analogue, which reduces the GA growth rate by 29% monthly and by 20% every other month (EOM) in phase II trial, compared with the sham treatment group (NCT02503332) [NCT03525613). Moreover, an extension study to assess the long-term safety and efficacy is currently under way (NCT03777332). Lastly, as a humanized IgG1 monoclonal antibody engineered to potently inhibit the activity of C3, the performance of NGM621 is safe and well-tolerated in phase I trials. Further studies will be conducted with doses of 15 mg injected every 4 weeks or every 8 weeks in phase II trial (NCT04465955).Targeting at C3, three therapeutic drugs have been devised for dry AMD so far. Firstly, POT-4, also referred to as AL-78898A, is a compstatin derivative, which is also the first complement inhibitor to be employed in patients. Preliminary results have shown that intravitreal POT-4 is safe and well-tolerated (2503332) . Of noteNCT00935883) [NCT01527500). Finally, Zimura, also known as avacincaptad pegol, is a pegylated RNA aptamer, which is a specific and potent inhibitor of C5. Compared with the sham group, intravitreal administration of Zimura 2 mg and 4 mg dose groups can reduce 27.4% and 27.8% of mean rate of GA growth over one year, respectively, which is of statistical significance in the phase II/III trials (NCT02686658) [NCT04435366).Targeting at C5, there are three therapeutic drugs explored for GA up to now. First, as the first FDA-approved complement inhibitor, eculizumab was safe and well-tolerated in the body through 6 months. However, eculizumab did not significantly reduce the growth rate of GA or the volume of drusen by intravenous fluid (0935883) , 112. Se2686658) . In addiNCT02288559) [NCT02247479, NCT02247531) [NCT01835015). Unfortunately, CLG561 were evaluated as a monotherapy or in combination with LFG316 (a C5 inhibitor), and there is no effect on the change of GA lesions in the phase II clinical trial (NCT02515942). Besides, as a novel drug, IONIS-FB-LRx is a ligand-conjugated antisense inhibitor, which can be administered subcutaneously for systemic reduction in circulating FB levels, with a potential to diminish the systemic overactivity of the alternative pathway. It is being assessed in a phase II trial for dry AMD patients. In phase I trials, subcutaneous injection of IONIS-FB-LRx has been proved safe in 54 healthy volunteers. Based on this, a phase II trial has been initiated to evaluate the effectiveness of IONIS-FB-LRx in GA patients (NCT03815825).On the one hand, several inhibiting activators have been targeted in clinical trials, such as FD, properdin, and FB. As is known to all, complement FD is a pivotal activator of complement AP, which is the target of lampalizumab, a selective monoclonal complement FD inhibitor. In phase II, a positive result indicated that monthly lampalizumab treatment demonstrates a 20% reduction in the progression of GA lesion area (2288559) . However2247531) , 116. OnNCT03144999). Of note, while the phase I trials are not designed to judge efficacy, it is encouraging that 9 of 11 cases demonstrated a slower rate of GA growth, which promotes it to be tested for the efficacy of GA in phase II trials (NCT04358471).On the other hand, a few supplementing regulators of the complement system have been targeted in clinical trials, for example, CD59, FI, and FH. Firstly, AAVCAGsCD59, also named as HMR59, uses an AAV2 gene therapy and is designed to induce the generation of a soluble CD59 protein, which binds the incomplete MAC and blocks the binding of the C9 protein required to complete the complex , 118. InNCT03846193). Moreover, gene therapy provides the potential for a single injection lasting for 1 year or longer, which offers the benefit of requiring fewer injections to treat AMD patients should the drug show efficacy and safety in future trials. As an endogenous human FH protein, GEM103 is administered via intravitreal injection. The safety and tolerability of GEM103 has been shown in phase I trials. Ocular pharmacokinetic and pharmacodynamic effects were assessed in aqueous humor samples for two months after treatment, showing that sustained supranormal levels of drug concentrations were achieved after treatment.Furthermore, two clinical trials based on genotype to select patients have been performed. Specifically, before the inclusion in the trials of GT005 and GEM103, patients are chosen for carrying risk gene variants in complement FI and FH. Similar to AAVCAGsCD59, GT005 uses an AAV vector and is designed to the supply of FI protein to the treated eye. Nowadays, phase I/II trials of GT005 in patients with GA are assessing its effectiveness in the UK. As therapy via subretinal injection, GT005 is currently in phase II clinical trials (NCT04656561).Finally, ANX007 is a monoclonal antibody antigen-binding fragment (Fab), which can potently bind to C1q to inhibit the activation of CP, including C3 and C5. ANX007 has been tested in patients with primary open-angle glaucoma, and the safety is confirmed, and it is well-tolerated in the eye. At present, ANX007 is used in investigating the safety and efficacy of intravitreal injections in patients with GA in phase II . The other is APL-2, which is delivered intravitreally and well-tolerated in phase I trials (NCT02461771). APL-2 were evaluated in patients with active wet AMD on anti-VEGF treatment in phase II as well. However, this clinical trial was terminated due to the ineffectiveness of treatment when 17 participants were collected (NCT03465709).Targeting at C3, two therapeutic drugs have been devised for wet AMD patients so far. The intravitreal delivery of POT-4 was compared to anti-VEGF in patients with active wet AMD in a phase II trial. As a result, the advantage of POT-4 over anti-VEGF drugs could not be demonstrated. Instead, the AMD participants treated with POT-4 had increased retinal thickness at week 4, while patients injected with anti-VEGF had decreased retinal thickness. POT-4 was unable to reduce central retinal thickness 12-week posttreatment, as seen with ranibizumab (NCT01535950) nor improvement in BCVA and macular thickness (NCT01624636). Zimura combination therapy with ranibizumab for wet AMD was safe and well-tolerated after 6 months of treatment in a phase II trial as well . Of note, in patients receiving monthly Zimura 2 mg in combination with anti-VEGF 0.5 mg, approximately 60% achieved visual acuity improvement greater than or equal to three lines, which was better than the results of anti-VEGF monotherapy.Targeting at C5, there are two therapeutic agents that have been explored in wet AMD to date. LFG316 drug was also assessed for efficacy in phase II trials, unfortunately, neither reduction in the number of anti-VEGF injections (NCT03585556). Previous studies have shown that subretinal injection of AAVCAGsCD59 attenuated the formation of laser-induced CNV by around 60% in mice [NCT03814291). The results from these clinical trials will provide insight into the potential of complement inhibition and combination therapy in the future [Moreover, two new drugs are undergoing phase I clinical trials. Firstly, intravitreal delivery of AAVCAGsCD59 is currently in phase I clinical evaluation . However, FAF has poor susceptibility to media opacities because of the macular pigment that absorbs blue light, thereby causing difficulty in imaging the fovea . TherefoAlthough complement therapy is confronted with multiple challenges currently, its therapeutic future remains promising. Both APL-2 and Zimura have demonstrated modest success in inhibiting the progress of GA. With the improvement of visual acuity as an ideal goal, both of them have passed phase II clinical trials and are undergoing further verification in phase III clinical trials. Of note, the inhibition of C3 through APL-2 induced neovascularization, prompting a tendency to convert dry AMD to wet AMD. Even though there remain unrevealed risks of neovascularization in dry AMD, treatment with anti-VEGF after conversion to wet AMD is also a possible treatment strategy for this group of patients . In addiThe complement system indeed plays a remarkable role in the development and progression of AMD. Targeting at different complement components, many clinical trials of complement inhibitors have been conducted or are ongoing in AMD. While most complement inhibitors fail to demonstrate the potential, APL-2 and Zimura are effective in inhibiting the rate of GA progression in phase II clinical trials, and both of them successfully entered phase III trials. Furthermore, the combination of Zimura and ranibizumab resulted in a significant improvement in visual acuity in patients with wet AMD at phase II clinical trials compared to ranibizumab monotherapy. Of note, the performance of Zimura in both dry AMD and wet AMD is quite encouraging. However, whether Zimura is expected to be a new drug for dry AMD is pending the results of phase III clinical trials. Overall, complement inhibitors have shown potential in the future treatment of AMD and deserve to be explored further.


Saccharomyces cerevisiae) culture (YC) for the enhancement of growth performance and general animal health. Grain-based pelleted total mixed rations (TMR) are emerging in intensive sheep farming systems, but it is uncertain if the process of pelleting results in YC becoming ineffective. This study aimed to examine the effects of YC supplemented to pelleted TMR at two proportions of corn in the diet on animal performance, feed digestion, blood parameters, rumen fermentation, and microbial community in fattening lambs. A 2 × 2 factorial design was adopted with two experimental factors and two levels in each factor, resulting in four treatments: (1) low proportion of corn in the diet  without YC, (2) LC with YC (5 g/kg diet), (3) high proportion of corn in the diet  without YC, and (4) HC with YC. Fifty-six 3-month-old male F2 hybrids of thin-tailed sheep and Northeast fine-wool sheep with a liveweight of 19.9 ± 2.7 kg were randomly assigned to the four treatment groups with an equal number of animals in each group. The results showed that live yeast cells could not survive during pelleting, and thus, any biological effects of the YC were the result of feeding dead yeast and the metabolites of yeast fermentation rather than live yeast cells. The supplementation of YC resulted in 31.1 g/day more average daily gain regardless of the proportion of corn in the diet with unchanged feed intake during the 56-day growth measurement period. The digestibility of neutral detergent fibre and acid detergent fibre was increased, but the digestibility of dry matter, organic matter, and crude protein was not affected by YC. The supplementation of YC altered the rumen bacterial population and species, but the most abundant phyla Bacteroidetes, Firmicutes, and Proteobacteria remained unchanged. This study indicates that YC products can be supplemented to pelleted TMR for improved lamb growth performance, although live yeast cells are inactive after pelleting. The improved performance could be attributed to improved fibre digestibility.There is a growing interest in the use of yeast ( Sheep is of economic importance in Northeast China . Sheep fOne of the common feed additives used by farmers and researchers is yeast culture (YC) , which iTo increase growth rates in sheep production, farmers tend to increase the proportion of cereal grains in the diet . HoweverFeeding total mixed rations (TMR) has been popular due to its advantages of providing balanced nutrients in the diet, the relatively stable rumen environment, reduced feed sorting, and better animal performance in dairy cows . TMR feePelleting is a critical step in the manufacture of pelleted TMR and involves steam conditioning, high temperature, and high pressure. These processes could cause changes to the nutritional value of the diet through chemical alterations  and coulWe hypothesise that YC supplemented to pelleted TMR still has beneficial effects on fattening lambs. The objective of the study was to examine the effects of supplementing YC to pelleted TMR containing two proportions of corn in the diet on animal performance, digestibility, blood metabolites, rumen fermentation parameters, and ruminal microbial communities of fattening lambs.Saccharomyces cerevisiae) culture (YC) supplemented (CON), (2) LC with YC supplemented at the 5 g/kg diet, (3) high proportion of corn in the diet  without YC, and (4) HC with YC supplemented at the 5 g/kg diet. The dose of YC at the 5 g/kg diet was recommended by the YC producer. The proportion of corn at the 350 g/kg diet was recommended by researchers  low proportion of corn in the diet  without yeast  (n = 14 per treatment).Sixty male F2 hybrid lambs of thin-tailed sheep and Northeast fine-wool sheep, the most popular breed in the region (1), aged 3 months with a liveweight of 19.9 ± 2.7 kg, were used in the experiment. They were de-wormed, examined twice for brucellosis using the Rose Bengal plate test (la spp.) , and theAll lambs were individually weighed fortnightly for 8 weeks. The weighing was conducted at the same time of the day before the morning feeding each time using an electronic scale for livestock with a precision of 0.05 kg . Average daily gain (ADG) was estimated using a plot of liveweight against time.On day 56 after weighing, blood samples were collected randomly from six lambs from each treatment before the morning feeding. On day 70, rumen samples were randomly collected before the morning feed from 12 lambs to characterise the rumen microbial profile. On the same day, six lambs were randomly removed from each treatment for a digestibility trial and another six lambs were removed for slaughter. At the end of the digestibility trial, rumen samples were taken on day 82 at 3 h after morning feeding on these lambs for the determination of fermentation characteristics. Slaughter was performed on days 71 and 72 with three lambs each day for each treatment group. The remaining two lambs were released from the experiment on day 70.The ingredients and nutrient contents of the experimental diets are presented in All lambs were kept in the same room with a ventilation fan that operated for 24 h a day. No bedding was provided, but the pens were cleaned every morning before feeding. Lambs were offered feed twice a day with equal proportions at 08:00 and 16:00 h. Feed allowance was such that 10–20% of the feed offered was not eaten. Feed offered and refused was quantified daily for the estimation of feed intake. Lambs were group fed with 14 in each pen, except during the digestibility measurement period when lambs were individually fed in metabolic crates. Water was available at all times.2SO4 and stirred twice daily to homogenise urine and acid. Urine volume was measured, and one-tenth was subsampled and pooled for each animal. Faeces and urine subsamples were stored at −20°C. Feed refusals were not subsampled daily but pooled for each animal. At the end of the trial, daily feed samples were pooled for each dietary treatment. Feed, refusal, and faeces were dried at 65°C for 48 h, ground to pass a 1-mm sieve and sealed in bottles for chemical composition analysis.The digestibility trial began on day 70 and was carried out over 12 days. Lambs were given 6 days for adaption and 6 days for sample collection. Feed offered and refused, and the outputs of faeces and urine were collected and quantified daily using the total faeces collection technique . One-fifca. 50 ml) for the analysis of rumen fermentation parameters were taken using an oesophageal tube with the first 50 ml discarded to minimise saliva contamination in a procedure described by Huo et al.  and then stored at −20°C.Rumen samples . Fresh organs were measured using an electronic scale with a precision of 1 g . Organ indexes were calculated as organ weights divided by live BW.After fasting for 24 h, lambs were weighed and slaughtered, and hot carcass weight (HCW), backfat thickness, loin eye area, and fresh organs were quantified. HCW was recorded with kidneys included. The dressing percentage was calculated as HCW divided by live body (BW) ×100. Backfat thickness was measured in the method of Kirton et al. . The loiThe live yeast cells in the YC products and diets were counted under a microscope after staining with alkaline methylene blue .Feed samples were determined for dry matter (DM) , ash 3434, CP  ruminantium group (P < 0.001), and increases in Gastranaerophilales_norank (P = 0.010) and Anaerobiospirillum (P = 0.033). Among these genera, the [Eubacterium] coprostanoligenes group (P = 0.021) and Ruminococcaceae NK4A214 group (P = 0.096) decreased, and Gastranaerophilales_norank (P = 0.035) increased with the supplementation of YC, cellulolytic bacteria Fibrobacter, Ruminococcus 1, and Butyrivibrio 2, and other bacteria were not significantly affected. Lactate-producing bacteria Lactobacillus, Streptococcus, and Bifidobacterium were < 0.1% in total bacteria and thus not listed in Megasphaera and Selenomonas 3 were < 1.2% and 0.5% in any one of the four treatment groups and not affected by YC supplementation.Increases in the corn proportion resulted in decreases in the abundance of the [Eubacterium] 8 cfu/g live yeast cells and was supplemented at 5 g/kg feed, which was expected to have 1 × 106 cfu/g live cells in the feed if all live yeast cells survived. However, the results from the microscopical evaluation showed that the number of live yeast cells was <10 × 103 cfu/g with a survival rate of <1%, which was the same as the CON, indicating that live yeast in the YC product was inactivated during the process of pelleting. Yeast is sensitive to high temperatures , as project PRJNA694322, submission ID SUB8925435. All the rest of the relevant data are within the paper and its supporting information file.Sequencing data are available in the NCBI Sequence Read Archive .XS, PY, HW, and KK conceived and planned the study. XS acquired funding, supervised all research, analysed and interpreted data, prepared the tables, prepared the figures, and wrote the manuscript. BS, TW, WY, MW, BL, YH, QH, CL, WT, RL, JL, CW, and XS collected data. XS and JB revised the manuscript. All authors contributed to the article and approved the submitted version.PY and WY were employed by Portal Agri-Industries Co., Ltd. HW and KK were employed by Angel Yeast Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders of the study were not involved in the study design, the collection, analysis, and interpretation of data, the writing of this article or the decision to submit it for publication.


The CH4 is originated from rumen fermentation and its concentration is affected by several factors, including genetics and nutrition. Ruminants have an extraordinary symbiosis with microorganisms  that ferment otherwise indigestible carbohydrates, from which they obtain energy to grow and continue actively producing, among other products, volatile fatty acids, CO2 and H2. Detrimental ruminal accumulation of H2 is avoided by methanogenesis carried out by Archaea methanogens. Importantly, methanogenesis is not the only H2 sink pathway. In fact, other bacteria can reduce substrates using metabolic hydrogen formed during carbohydrate fermentation, namely propionate production and reductive acetogenesis, thus lowering the CH4 produced. Although the complexity of rumen poses challenges to mitigate CH4 production, the emergence of sequencing techniques that allow the study of microbial communities, gene expression, and metabolome are largely contributing to unravel pathways and key players in the rumen. Indeed, it is now recognized that in vivo emissions of CH4 are correlated to microbial communities, and particularly with the abundance of methanogens, several bacterial groups, and  their genes. The goal of CH4 mitigation is to work in favor of the natural processes, without compromising rumen function, animal health, and productivity. Notwithstanding, the major challenge continues to be the feasibility and affordability of the proposed solutions.Agriculture is responsible for a great share of the anthropogenic sources of greenhouse gases that, by warming the earth, threaten its biodiversity. Among greenhouse gas emissions, enteric CH Th. Th96]. inantium  and Succccinate) . Accordin for H2 . Similare of CH4 . Another thoenin . Lactiplopionate . In turnmissions . This himissions .Methanobrevibacter over Methanomassiliicoccus, and a successful shift of H2 flow towards the propionate pathway [Manipulation of fiber content, namely by the replacement of forage fiber by non-forage fiber sources, promoted an expansion of Firmicutes over Bacteroidetes and of  pathway . Further pathway .Succinivibrio (family Succinivibrionaceae), Roseburia, and Blautia (family Lachnospiraceae) with the increase of propionate, when testing diets that differed in the corn processing techniques [Succinivibrionaceae were effectively associated with high propionate and low CH4 yield [In vivo studies associated other bacteria such as genera chniques . FurtherH4 yield , 108.Adding to its potential as a methanogenesis competitor, the increase of propionate production is advantageous for cows’ health and efficiency. The major share (50 – 75%) of propionate produced is absorbed into the portal vein . Propion2 and four moles of H2 are incorporated per one mole of acetate produced (G), in a thermodynamically feasible set of reactions.(G).Acetogenesis has been documented as an alternative to methanogenesis, in which via the acetyl-CoA pathway, two moles of CO2, and in the rumen of cows and sheep, the dominant microorganisms are members of Lachnospiraceae, Clostridiaceae, and Ruminococcaceae families [2 utilization is lower for methanogens than it is for acetogens, which renders reductive acetogenesis a disadvantage. Therefore, increasing H2 pressure would remove methanogenesis thermodynamic advantage [Eubacterium limosum was able to produce acetate when methanogens were suppressed concomitantly, but a minimal change in CH4 production was observed when methanogenesis was not inhibited [E. limosum and Proteiniphilum acetatigenes decreased CH4 concentrations, increased acetate, whereas in vivo, P. acetatigenes was associated with high milk protein, lower somatic cell counts, and lower decline of milk production over 60 days [Acetogens degrade multiple substrates  in addition to Hfamilies , 113. Thdvantage . An in vnhibited . Similar 60 days .4 emitting animals. That knowledge has recently been prompted by metagenomics and transcriptomics that allow not only studying the rumen microbial composition but also its function, unraveling genes and pathways involved in the metabolism of H2. The goal of successful interventions is likely shifting H2 from methanogenesis, exploiting natural processes, without compromising rumen physiology. That depends upon the interaction between microbial communities, including several orders of bacteria, archaea, and eukaryotes that encode and express enzymes [2 that can be utilized by acetogens, though a combined strategy to reduce methanogens is required.Several in vivo studies employing sequencing techniques have revealed distinct ruminal microbial communities between high- and low-CH enzymes , thus me4 emissions, thus addressing the role of cattle in the current climate emergency.Even though knowledge on the ruminal microbiome has expanded, there are still several genes and microorganisms whose characterization and function remains unknown, particularly protozoan and fungi. Recent works have recovered archaeal and bacterial MAGs from metagenomic data, from which new, previously unnoticed, pathways and networks have been discovered. This indicates that the ruminal microbiome study is a work in progress and may provide us new prospects for finding solutions for lowering livestock enteric CH