Lipopolysaccharide (LPS) proved to be an important tool, not only in the induction of neuroinflammatory models, but also in demonstrating the behavioral and cognitive consequences of endotoxemia. Curcumin, in its native form, has proven to be a worthy candidate for further development as it protects the dopaminergic neurons against LPS-induced neurotoxicity. However, it remains hindered by its poor bioavailability. In this study we aim to explore the possible molecular mechanism of LPS-induced neurotoxicity and the possible protective effects of orally supplemented nanocurcumin. Thirty-six adult male Wistar rats weighing 170–175 g were divided into six groups and treated with single I.P.  dose of LPS  (5 mg/kg BW) plus daily oral nanocurcumin (15 mg/kg BW). The rats were followed for 7 days after the LPS injection and nanocurcumin supplementations daily via oral gavage. After scarification, the levels of neurotransmitters, antioxidants, and amyloidogenesis markers were assessed in brain tissues. Nanocurcumin showed adequate antioxidant and neuroprotective effects, rescuing the rats which had been injected intraperitoneally with LPS endotoxin. E. coli and Salmonella sp. outer membranes [Lipopolysaccharide (LPS) is the most common glycolipid on the cell surface of most Gram-negative bacteria, accounting for up to 80% of embranes . It is pembranes . It is uembranes .LPS proved to be an important tool not only in the induction of neuroinflammatory models  but also+2, trichloroacetic acid extraction at 4C, and extraction in water at 100C. For rough LPS purification with chloroform, petroleum ether, phenol, and methanol may be applied [Escherichia coli Gram-negative bacteria [The amphipathic nature of LPS proved to be an obstacle in its extraction from Gram-negative bacteria . Many is applied . For smo applied . Westphabacteria .LPS induces its neuroinflammatory response in mammals by binding to toll-like receptor 4 (TLR-4), which is a pattern recognition receptor (PRR) that is a critical immune response driver to bacterial infections . InterleCurcumin, in its native form, has proven to be a worthy candidate for further development, as it protects the dopaminergic neurons against LPS-induced neurotoxicity . HindereThe challenges associated with water-insoluble molecules have been resolved using nanotechnology . The nanCurcumin nanoformulations have dramatically transformed how diseases are treated by improving bioavailability, cellular absorption, and permeability with increased plasma concentration . For theUtilizing the tools provided by the field of nanotechnology to deliver nanocurcumin particles instead of the native curcumin provided a better solubility and bioavailability. It also proved to be a more effective alternative for native curcumin and it has been reported that the nanoform of curcumin is the better form for preventing neurodegenerative changes in experimentally induced cerebral malaria . NanocurThis study aims to explore the possible molecular mechanism of LPS-induced neurotoxicity and the possible protective effects of orally supplemented nanocurcumin suspension. This work was accomplished through the assessment of cerebral cortex and hippocampal changes at different levels, including histological, neurotransmitter, amyloidogenic pathway, redox and antioxidant status, inflammatory status, and gene expression of the mitochondrial transcription factor (mTFA).Escherichia coli O111:B4, Product Number L2630, Sigma-Aldrich Chemie GmbH Eschenstrasse 5 D-82024 TAUFKIRCHEN company). The extracted LPS was obtained from E. coli ATCC 25922 bacteria according to El-Moslemany et al. [Sigma lipopolysaccharides (LPSs) were purchased from Sigma Aldrich, Cedex, France  were centrifuged at 9000× g for 8 min. The pellets were washed twice in PBS  containing 0.15 mM CaCl2 and 0.5 mM MgCl2. Bacterial pellets were resuspended in 5 mL PBS and sonicated for 10 min on ice. To eliminate contaminating proteins and nucleic acids, treatment with proteinase K, DNase, and RNase was performed. Proteinase K (100 μg/mL)  was added to the bacterial suspensions and the tubes were kept at 65 °C for one hour. The mixture was subsequently treated with RNase (40 μg/mL)  and DNase (20 μg/mL)  in the presence of 1 μL/mL 20% MgSO4 and 4 μL/mL chloroform. Incubation continued at 37 °C overnight. At the next step, an equal volume of hot (65–70 °C) 90% phenol was added to the mixtures, followed by shaking at 65–70 °C for 15 min. Suspensions were then cooled on ice, transferred to 1.5 mL tubes, and centrifuged at 9000× g for 15 min. Supernatants were transferred to 15 mL conical centrifuge tubes and phenol phases were re-extracted by 300 μL distilled water. Sodium acetate at 0.5 M final concentration and 10 volumes of 95% ethanol were added to the extracts. Samples were stored at −40 °C overnight in order to precipitate LPS. Tubes were then centrifuged at 4000× g 4 °C for 15 min and the pellets were suspended in 1 mL distilled water. Extensive dialysis against double-distilled water at 4 °C was carried out at the next step until the residual phenol in the aqueous phases was totally eliminated. Final purified LPS product was lyophilized and stored at 4 °C.Lipopolysaccharide was extracted by hot phenol–water method, as described previously, with some modifications . In brieAs per the company of origin (Nanotech Egypt for photo-electronics) of the purchased nanocurcumin, the nanocurcumin powder was prepared by emulsification–solvent evaporation using Annaraj et al.’s method . Bulk cuPVA-loaded nanocurcumin thus obtained was characterized by UV-Vis absorption spectra that was obtained on an Ocean Optics USB2000+VIS-NIR Fiber optics spectrophotometer. Furthermore, to check the shape and size, TEM was performed on JEOL JEM-2100  high-resolution transmission electron microscope at an accelerating voltage of 200 kV. The obtained nanocurcumin had a spherical-like shape, with a particle size of 50 ± 5.5 nm.Thirty-six adult male Wistar rats aged 12–14 weeks and weighing 170–175 g were obtained from the animal facility unit, Faculty of Medicine, Alexandria University, Alexandria, Egypt. Rats were housed at a controlled room temperature (25 °C) with a natural light–dark cycle (12/12 h) for two weeks for acclimation, with diet and water ad libitum throughout the experimental period. All animal procedures and experimental protocols were carried out in accordance with the Alexandria University Institutional Animal Care and Use Committee (IACUC) guidelines (AU14-200922-1-9E). During the experiment, every effort was made to reduce the rats’ suffering.The rats were divided into 6 groups (6 rats each). Control group: Healthy rats that received no supplements. Nanocurcumin-treated group: The rats were daily treated orally by gavage with nanocurcumin (15 mg/kg BW). Extracted LPS-treated rats: The rats were treated with single I.P. dose of extracted LPS (5 mg/kg BW). Extracted LPS+nanocurcumin rats: The rats were treated with single I.P. dose of extracted LPS (5 mg/kg BW) plus daily oral nanocurcumin (15 mg/Kg BW). Sigma LPS-treated rats: The rats were treated with single I.P. dose of Sigma LPS (5 mg/kg BW). Sigma LPS+nanocurcumin rats: The rats were treated with single I.P. dose of sigma LPS (5 mg/kg BW) plus daily oral nanocurcumin (15 mg/kg BW). The rats were followed for 7 days after the LPS injection and nanocurcumin supplementations daily via oral gavage .g for 20 min at 4 °C and the supernatants were stored at −80 °C for further analysis. At the end of the experimental period, all animals were anesthetized with isoflurane inhalation and sacrificed by cervical dislocation. The brain was rapidly removed, washed with (0.9%) saline solution, and cleaned of adhering matters. The two hemispheres were separated; one hemisphere was fixed in normal formalin for the histological study and the second hemisphere was used to obtain the cerebral cortex and hippocampus. The cerebral cortex and hippocampus were divided into two parts: One was used for total RNA isolation for gene expression analysis, and the second part was homogenized in ice-cold phosphate buffered saline (1:9). The homogenates were centrifuged at 10000× The tissue levels of acetylcholine (Ach), dopamine (DA), serotonin, and acetylcholine esterase were assayed using specific ELISA kits , according to the manufacturer’s instructions.Malondialdehyde in the whole homogenate of the cerebral cortex and hippocampus were determined according to the method from Draper and Hadley . The sam20, 0.1 M HCl, and 0.1 M NH4OH (pH 9.6). The deproteinized samples (by ZnSO4 precipitation) were added to the activated Cd beads and incubated at room temperature overnight with agitation. After centrifugation, 100 μL of the supernatants was added into duplicate wells, then 50 μL of sulfanilamide solution was added and shaken briefly. An amount of 50 μL of N-(1-Naphthyl) ethylenediamine dihydrochloride,  was added and shaken for 5 min at room temperature. Then, the absorbance values at 540 nm was read in a microtiter plate reader .The nitrite and nitrate (named NOx) concentration was determined by simple Griess reaction . BecauseThe standard curve was plotted and the concentrations of the samples were estimated from the curve.The enzymatic method described by Griffith et al. was used to measure the total glutathione and GSSG content . This isThe tissue levels of a TNF-α and Aβ1-42 were assayed using specific ELISA kits  according to the manufacturer’s instructions. Both kits use the quantitative sandwich enzyme immunoassay technique. Antibody specific for TNF-α or Aβ1-42 was precoated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α or Aβ1-42 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for TNF-α or Aβ1-42 is added to the wells. After washing, avidin-conjugated horseradish peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TNF-α or Aβ1-42 bound in the initial step. The color development is stopped, and the intensity of the color is measured using a microplate reader .pNA substrate were added. The plate was covered and incubated at 37 °C for 2 h. The absorbance in the wells was measured at 405 nm using a microplate reader . The pNA standard curve was constructed by diluting the pNA stock solution (10 mM) in DMSO to prepare serial dilutions between 0 and 100 pmol/µL.Caspase-3 has a specificity for cleavage at the terminal side of aspartate residue of the amino acid sequence DEVD . The caspase-3 enzymatic activity was assayed using Caspase-3 Assay Kit . This kit is used to conjugate caspase-3 sequence-specific peptide acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) to yellow group p-nitroaniline (pNA). When the substrate is cut by caspase-3, the yellow group pNA is dissociated. pNA has an absorption peak at 405 nm. The OD value was measured at 405 nm and then the caspase-3 activity was indirectly calculated. The assay was performed in a total volume of 100 µL in a 96-well plate and the absorbance was read using a plate reader. To 50 µL of the tissue homogenate supernatant, 40 µL of caspase-3 assay buffer and 10 µL of Ac-DEVD-®, Hilden, Germany) and the extracted RNA was reverse-transcribed by QuantiTect Reverse Transcription Kit  following the manufacturer instructions. Then, complementary DNA (cDNA) were amplified and detected using specific primers .Quantitative analysis of BDNF, mTFA, BACE1, and Nrf2 expression were performed using two-step quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) based on SYBR-Green I fluorescence. First, the total RNA was isolated from the tissues with the RNeasy Mini Kit . The Shapiro–Wilk test was used to verify the normality of distribution. The data were expressed as mean ± standard deviation (SD). Comparisons between different groups were made using one-way ANOVA followed by Tukey’s post hoc test. Significance of the obtained results was judged at the 5% level.The results of the brain neurotransmitters of the studied groups are shown in In the cortex, the control rats supplemented with nanocurcumin showed a significant increase in the level of Ach compared with the control rats. The rats treated with LPS  showed significantly lower Ach levels than the control rats. The cosupplementation of the LPS-treated rats with nanocurcumin significantly increased the cortical content of Ach compared with unsupplemented LPS-treated rats . There was no significant difference between the rats treated with extracted LPS compared with the rats treated with commercial LPS .In the hippocampus, the supplementation of normal rats with nanocurcumin showed no significant effects on the Ach level. The rats treated with LPS  showed significantly lower levels of Ach compared with the control rats. The cosupplementation of the LPS-treated rats  with nanocurcumin significantly increased the hippocampal content of Ach compared to the unsupplemented LPS-treated rats . There was no significant difference between the rats treated with extracted LPS compared with the rats treated with commercial LPS .In the cortex, supplementation with nanocurcumin significantly increased the AchE content in the control rats. The rats treated with LPS  showed significantly higher content of AchE compared with the control rats or rats supplemented with nanocurcumin. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of AchE compared with the unsupplemented LPS-treated rats .In the hippocampus, the rats treated with LPS  showed significantly higher content of AchE compared with the control rats. Those rats treated with the commercial LPS had significantly higher AchE content than the extracted LPS-treated rats. The cosupplementation of the extracted LPS-treated rats with nanocurcumin significantly normalized the cortical content of AchE, while the supplementation of commercial LPS-treated rats with nanocurcumin significantly reduced the AchE content compared with unsupplemented rats, but was still significantly higher than the other groups .In the cortex, the control rats supplemented with nanocurcumin showed a significant increase in the level of serotonin compared with the control rats. The rats treated with LPS  showed significantly lower levels of serotonin than the control rats, with no significant difference between the extracted and commercial LPS. The cosupplementation of LPS-treated rats  with nanocurcumin significantly normalized the cortical content of serotonin compared with unsupplemented LPS-treated rats  .In the hippocampus, the rats treated with extracted LPS showed mild but significantly lower serotonin levels, while those treated with commercial LPS showed a marked decline in the serotonin levels compared with the control rats. The cosupplementation of LPS-treated rats  with nanocurcumin did not significantly affect the hippocampal content of serotonin compared with unsupplemented LPS-treated rats .In the cortex, the control rats supplemented with nanocurcumin showed a nonsignificant decline in the level of dopamine compared with the control rats. The rats treated with LPS  showed significantly lower levels of dopamine compared with the control rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly increased the cortical content of dopamine compared with unsupplemented extracted LPS-treated rats and completely normalized in extracted LPS-treated rats .In the hippocampus, no significant difference was observed in the dopamine content between all studied groups .In the cortex, the control rats supplemented with nanocurcumin showed a nonsignificant increase in the expression of BDNF compared with the control rats. The LPS-treated rats  had significantly lower BDNF expression compared with the control rats. The LPS-treated rats  cosupplemented with nanocurcumin showed completely normalized NRF2 expression .In the hippocampus, the control rats supplemented with nanocurcumin showed no significant change in the BDNF expression compared with the control rats. The rats treated with extracted LPS (cosupplemented or unsupplemented with nanocurcumin) showed no significant change in the BDNF expression compared with the control rats. On the other hand, the rats treated with the commercial LPS showed a significant reduction in BDNF gene expression compared with the control groups. The cosupplementation of the commercial LPS-treated rats with nanocurcumin significantly increased the BDNF expression compared with the unsupplemented rats .The control rats supplemented with nanocurcumin showed no significant difference in the level of total GSH compared with the control rats. In addition, all other groups of rats showed no significant changes in the levels of total glutathione compared with the control rats .Regarding the level of reduced glutathione (GSH), the rats treated with LPS  showed significantly lower cortical GSH levels compared with the control rats. The cosupplementation of LPS-treated rats with nanocurcumin increased the level of GSH compared with the unsupplemented rats; however, these increases are not significant .The rats treated with LPS  showed significantly higher levels of GSSG compared with the control rats. The commercial LPS-treated rats had significantly higher GSSG levels compared with the extracted LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of GSSG compared with unsupplemented LPS-treated rats . The rats treated with extracted LPS cosupplemented with nanocurcumin had significantly lower GSSG compared with the commercial LPS-treated rats cosupplemented with nanocurcumin .The redox ratio (GSH/GSSG) in the cortex combined the changes in GSH and GSSG. The ratio was significantly higher in the control rats supplemented with nanocurcumin compared with the control rats. The rats treated with LPS  had a marked reduction in the GSH/GSSG ratio compared with the control group, with no significant difference between the extracted and commercial LPS. The cosupplementation of the LPS-treated rats  with nanocurcumin significantly raised the cortical GSH/GSSG ratio compared with unsupplemented LPS-treated rats .The control rats supplemented with nanocurcumin showed no significant difference in the level of GSH compared with the control rats. The rats treated with LPS  showed significantly lower total GSH levels compared with the control rats. The cosupplementation of LPS-treated rats does not significantly affect the total GSH levels compared with the unsupplemented rats .The levels of reduced GSH) are significantly lower in rats treated with LPS  compared with the control rats with the lowest level detected in the commercial LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly increased the level of GSH compared with the unsupplemented .The rats treated with LPS  showed significantly higher levels of GSSG compared with the control rats, the commercial LPS-treated rats have significantly higher GSSG levels compared with the extracted LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly declined the hippocampal content of GSSG compared with unsupplemented LPS-treated rats. The rats extracted LPS-treated cosupplemented with nanocurcumin have near the normal level and significantly lower GSSG compared with the commercial LPS-treated rats cosupplemented with nanocurcumin .The rats treated with LPS  have a marked reduction in the GSH/GSSG ratio compared with the control group with no significant difference between the extracted and commercial LPS. The cosupplementation of LPS-treated rats  with nanocurcumin significantly raise the cortical GSH/GSSG ratio compared with unsupplemented LPS-treated rats .In the cortex, the LPS-treated rats  have lower NRF2 expression levels. These effects are not significant compared with the control rats. In addition, the rats treated with the extracted or commercial LPS cosupplemented with nanocurcumin showed significantly higher expressions of NRF2 compared with the unsupplemented rats .In the hippocampus, the rats treated with extracted LPS showed significant suppression of the NRF2 expression compared with the control rats and the cosupplementation of these rats with nanocurcumin significantly normalized the expression. On the other hand, the rats treated with the commercial LPS showed a significantly marked reduction in the NRF2 gene expression compared with the control groups. The cosupplementation of the commercial LPS-treated rats with nanocurcumin significantly increased the NRF2 expression compared with the unsupplemented rats .The In the cortex, the rats treated with LPS  showed significantly higher levels of MDA compared with the control rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of MDA compared with unsupplemented LPS-treated rats . There was no significant difference between the rats treated with commercial LPS compared with the rats treated with extracted LPS .In the hippocampus, the rats treated with LPS  showed significantly higher levels of MDA compared with the control rats. The rats treated with commercial LPS had significantly higher MDA levels compared with the rats treated with extracted LPS. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the hippocampal content of MDA compared with unsupplemented LPS-treated rats  .In the cortex, the LPS-treated rats  showed significantly higher levels of NOx compared with the control rats, with the highest level of NOx detected in commercial LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of NOx compared with unsupplemented LPS-treated rats . There was a significant increase in levels of NOx in the rats treated with commercial LPS compared with the rats treated with extracted LPS .In the hippocampus, the rats treated with LPS  showed significantly higher levels of NOx compared with the control rats, with the highest level of NOx detected in commercial LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the hippocampal content of NOx compared with unsupplemented LPS-treated rats  .In the cortex, the rats treated with LPS  showed significantly higher BACE1 expression compared with the control rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly normalized the cortical expression of BACE1 compared with unsupplemented LPS-treated rats . There was no significant difference between the rats treated with extracted LPS compared with the rats treated with commercial LPS .In the hippocampus, the expression of BACE1 showed no significant changes between the studied groups .In the cortex, the rats treated with LPS  showed significantly higher levels of AB 1-42 compared with the control rats. The supplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of AB 1-42 compared with unsupplemented LPS-treated rats . There was no significant difference between the rats treated with extracted LPS compared with the rats treated with commercial LPS .In the hippocampus, the rats treated with LPS  showed significantly higher levels of AB 1-42 compared with the control rats. The rats treated with commercial LPS had a significantly higher level of AB 1-42 compared with those treated with the extracted LPS. The supplementation of LPS-treated rats  with nanocurcumin did not significantly affect the hippocampal content of AB 1-42 compared with unsupplemented LPS-treated rats  .In the cortex, the rats treated with LPS  showed significantly higher levels of TNF-α compared with the control rats. The commercial LPS-treated rats had significantly higher TNF- α levels compared with the extracted LPS-treated rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical content of TNF-α compared with unsupplemented LPS-treated rats . The rats treated with extracted LPS cosupplemented with nanocurcumin had significantly lower TNF-α compared with the commercial LPS-treated rats cosupplemented with nanocurcumin .In the hippocampus, the rats treated with extracted LPS showed significantly higher TNF-α levels by about 112%, while those treated with commercial LPS showed significantly higher TNF-α levels by about 417% compared with the control rats. The rats treated with commercial LPS had marked TNF-α levels compared with those treated with the extracted LPS. The supplementation of LPS-treated rats  with nanocurcumin caused a significant decline in the hippocampal content of TNF-α compared with unsupplemented LPS-treated rats . However, the commercial LPS-treated rats cosupplemented with nanocurcumin still had significantly higher marked TNF-α levels than the other groups .In the cortex, the rats treated with LPS  showed significantly higher activities of caspase-3 compared with the control rats. The cosupplementation of LPS-treated rats  with nanocurcumin significantly reduced the cortical activities of caspase-3 compared with unsupplemented LPS-treated rats . There was no significant difference between the rats treated with extracted LPS compared with the rats treated with commercial LPS .In the hippocampus, the rats treated with extracted LPS (supplemented or unsupplemented with nanocurcumin) showed significantly higher activities of caspase-3 compared with the control rats. Those rats treated with the commercial LPS had significantly higher activity compared with those treated with the extracted one. The cosupplementation of the LPS-treated rats with nanocurcumin showed significantly lower caspase-3 activity compared with the unsupplemented rats .In the cortex, the LPS-treated rats  had lower mTFA expression levels. These effects are not significant compared with the control rats. In addition, the rats treated with the extracted or commercial LPS and cosupplemented with nanocurcumin showed no significant effects on the expression of mTFA compared with the unsupplemented rats .In the hippocampus, the rats treated with extracted LPS showed significant suppression of the mTFA expression compared with the control rats and the cosupplementation of these rats with nanocurcumin showed no significant effect compared with the unsupplemented rats. On the other hand, the rats treated with the commercial LPS showed significantly reduced marked mTFA expression compared with the other groups. The cosupplementation of the commercial LPS-treated rats with nanocurcumin did not significantly affect the mTFA expression compared with the unsupplemented rats .At the histological level, the exposure to LPS did not induce any marked changes in the histological structure of the neurons or nerve fibers in the cerebral cortex .E. coli ATCC 25922 and examined its neurotoxic effects on the brains of male rats compared with the commercially obtained LPS. Both types of LPS induced similar neurotoxic effects through the induction of histological changes, neurotransmitter disturbances, induction of the amyloidogenic pathway, shifting the redox status, and the induction of inflammation, oxidative stress, and apoptosis in both the cerebral cortex and the hippocampus. Such changes should be associated with a behavioral change; however, we did not perform such behavioral tests in the present study. The understandings of the neurotoxic mechanisms of LPSs are mandatory to identify the most proper methods of protection against LPS neurotoxicity. This study demonstrates the potential protective role of orally administrated nanocurcumin against the LPS-induced neurotoxicity.In our lab, we successfully extracted the LPS from At the histological level, the exposure to LPS did not induce marked changes in the histological structure of neurons or nerve fibers. Soudi et al. showed in their preclinical study that, at a dose four-times the dose administered in this study, a moderate infiltration of inflammatory cells in the brain tissue was observed along with one aggregate in the meninges and congestion of the choroid plexus .At the neurotransmitter level, a single I.P. injection of both types of LPS  caused a significant decline in the levels of ACh, serotonin, and dopamine in both the cerebral cortex and the hippocampus. The status was worse in the rats treated with the commercial LPS than those treated with the extracted one. These abnormalities are associated with a significant increase in the tissue content of the AchE protein. The cosupplementation of LPS-treated rats with daily oral nanocurcumin significantly ameliorated the levels of neurotransmitters and the AchE protein content in the brain tissues, specifically in the cerebral cortex. Tyagi et al. observed increased AchE activity in their study, along with a marked increase in oxidative stress and an acute inflammatory response in the brain regions linked with several neurodegenerative diseases . In the The effect of LPS on the serotonergic system is well established and was demonstrated through an increase in the proinflammatory cytokines, causing anhedonia and a sickness behavior in the rats receiving I.P. LPS . FurtherThe neurotransmitter changes induced by LPS were associated with a significant decline in the cortex and hippocampus expressions of brain-derived neurotropic factor (BDNF). BDNF plays an important role in the activation of neurogenesis and the suppression of apoptosis, along with the modulation of synaptic activity. Thus, BDNF has an important role in several neurological diseases . Guan etIn accordance with our data, suppressed BDNF is associated with neurodegenerative diseases with neuronal loss, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and bipolar disease . Oral na1-42. These abnormalities are more prominent in the cerebral cortex than in the hippocampus. Moreover, the commercial LPS was a more powerful inducer of Aβ1-42 accumulation than the extracted LPS BACE1 code for β-secretase enzyme, which initiates the cleavage of amyloid precursor protein (APP) at the β-secretase site to produce amyloid beta-protein 1-42 (Aβ1-42) [1-42, demonstrating a high load of AB plaques and an increased risk of developing Alzheimer’s [1-42 were significantly reduced in the cortex of rats treated with both types of LPS. In the hippocampus, there was no significant ameliorating effect of the cosupplementation with nanocurcumin.Another important pathway that is affected by LPS is the amyloidogenic pathway. LPS caused marked induction in the expression of BACE1, which is associated with the marked elevation of Aβ(Aβ1-42) . The actheimer’s . After nRegarding neuroinflammation, LPS-injected rats showed marked elevation in the levels of the proinflammatory cytokine, TNF-α, especially in the rats treated with the commercial LPS. The highest elevation in TNF-α was observed in the hippocampus of rats treated with commercial LPS. It has also been shown that TNF-α is involved in enhancing neutrophil recruitment into sites of inflammation. The increase in TNF-α levels after a single I.P. injection of LPS into rats was previously reported by Bossu et al. . NanocurAt the redox level, both types of LPS induced significant reductions in the two main antioxidant systems in the brain: The glutathione (GSH) system and the nuclear factor erythroid 2–related factor 2 (NRF2) system. The levels of the reduced GSH were markedly depleted, while the level of the oxidized (GSSG) was significantly elevated, which caused a marked reduction in the redox ratio (GSH/GSSG) in the cerebral cortex and hippocampus of the LPS-treated rats. In addition, the cortex and hippocampus expression of NRF2 was significantly suppressed—especially in the hippocampus. These abnormalities in the redox systems were more prominent with the commercial LPS than the extracted one. Although the increase was not significant in the levels of total and reduced GSH after nanocurcumin supplementation, the level of GSSG significantly decreased, leading to a significant increase in the GSH/GSSG ratio in the cortical tissue. Furthermore, similar results were obtained in the hippocampal tissue. Being essential for the removal of the toxic ROS in the brain, the glutathione system plays a significant role in the protection of the brain tissue from the damage induced by oxidative stress . The impThe observed LPS depletion of the antioxidant systems was associated with marked elevation in the levels of malondialdehydes (MDA), the marker of lipid peroxidation and nitric oxide end products (NOx) in both cortex and hippocampus tissues. Together, impaired antioxidants and increased reactive oxygen species (ROS) production exacerbates the oxidative stress in brain tissues by LPS treatments. The elevated levels of MDA may be due to the overproduction of ROS induced by LPS, which was initially faced by the glutathione, and which results in the depletion of reduced GSH and elevation of GSSG. The disrupted redox status and induced oxidative stress of the brain tissues are associated with the induction of neuroinflammation and apoptosis, as indicated by the marked elevation of TNF-α levels and caspase-3 activity in the brain cortex and hippocampus. The rats receiving cosupplementation with nanocurcumin showed a marked decline in the levels of MDA, NOx, and caspase-3 activity. These effects are similar across the cortex and hippocampus. Rajasekar and colleagues obtained similar results with MDA in rats after supplementation with nanocurcumin crystals . In addiIn addition to being an apoptotic marker, caspase-3 has an important role in the process of normal brain development through the formation of apoptotic bodies . NanocurAt the molecular level, LPS significantly suppressed the gene expression of mitochondrial transcription factor A (mTFA), a transcriptional factor controlling the mitochondrial biogenesis in brain tissues through maintaining the copy number and structure of the mitochondrial DNA (mtDNA), as well as the transcription and replication of the mtDNA . MitochoThe mechanism by which LPSs suppress the brain gene expression of mtTFA is unclear. However, we can postulate that LPSs may have the ability to pass through the BBB to reach neuron cells, causing a direct consequence of oxidative stress and increased free radicals and lipid peroxide, which is confirmed in this study. The cosupplementation of LPS-treated rats with nanocurcumin improved the brain expression of the mTFA gene, which may completely normalize the levels in the cortex and hippocampus of the extracted LPS-treated rats and the cortex only in commercial-LPS treated rats, which may imply restoring mitochondrial biogenesis and function.Collectively, the extracted LPS and the commercially obtained LPS (Sigma Aldrich) showed nearly similar effects on most of the studied parameters, aside from some instances where the commercially obtained LPS produced a more pronounced effect. This difference in outcome between the two sources of LPS could be related to a difference in the species from which the LPS was isolated, since the O-antigen segment is different in the LPS extracted from any bacteria, even within the same species .In general, nanocurcumin particles showed neuroprotective and antioxidant effects, rescuing rats injected intraperitoneally with LPS endotoxin.LPSs have potential neurotoxic effects that significantly impact brain neurotransmitters, antioxidant systems, and mitochondrial biogenesis, and elicit neuroinflammation and apoptosis, which may increase the risk of neurological diseases. The study confirmed the protective effects of oral nanocurcumin against the LPS-induced neurotoxicity in the cerebral cortex and hippocampus, and recommends the application of nanocurcumin for the protection and/or treatment against septicemia-induced neurotoxicity.


Finally, we investigated the propagation of non-additive gene action in GRNs.Plant domestication is a remarkable example of rapid phenotypic transformation of polygenic traits, such as organ size. Evidence from a handful of study cases suggests this transformation is due to gene regulatory changes that result in non-additive phenotypes. Employing data from published genetic crosses, we estimated the role of non-additive gene action in the modulation of transcriptional landscapes in three domesticated plants: maize, sunflower, and chili pepper. Using A. We compared crosses between domesticated plants and their wild relatives to a set of control crosses that included a pair of subspecies evolving under natural selection and a set of inbred lines evolving under domestication. We found abundance differences on a higher portion of transcripts in crosses between domesticated-wild plants relative to the control crosses. These transcripts showed non-additive gene action more often in crosses of domesticated-wild plants than in our control crosses. This pattern was strong for genes associated with cell cycle and cell fate determination, which control organ size. We found weak but significant negative correlations between the number of targets of trans-acting genes (Out-degree) and both the magnitude of transcript abundance difference a well as the absolute degree of dominance. Likewise, we found that the number of regulators that control a gene’s expression (In-degree) is weakly but negatively correlated with the magnitude of transcript abundance differences. We observed that dominant-recessive gene action is highly propagable through GRNs. Finally, we found that transgressive gene action is driven by trans-acting regulators showing additive gene action.Our study highlights the role of non-additive gene action on modulating domestication-related traits, such as organ size via regulatory divergence. We propose that GRNs are shaped by regulatory changes at genes with modest connectivity, which reduces the effects of antagonistic pleiotropy. Finally, we provide empirical evidence of the propagation of non-additive gene action in GRNs, which suggests a transcriptional epistatic model for the control of polygenic traits, such as organ size.The online version contains supplementary material available at 10.1186/s13227-022-00206-4. Plant domestication has been widely employed as a model to study phenotypic evolution . One of Genes coding for cell cycle and cell fate determination are among relevant members of GRNs controlling meristem and, therefore, organ size, as they control the rates of cell division and cell expansion . It is lDrosophila and yeast that have assessed the association between GRN connectivity properties and transcript abundance differences, no studies have approached such associations in plant models.In diploid organisms, cis-acting mutations often produce additive gene action. A mechanistic explanation for this pattern is that cis-acting mutations act in an allele-specific fashion, so the expression of one allele does not affect the expression of the other and the resulting phenotype is the average of the parental alleles. Conversely, trans-acting factors bind to cis-regulatory elements of the two alleles, modulating the phenotype towards one parent, and thus producing non-additive (dominant-recessive) gene action , 17. As Zea mays), chili pepper (Capsicum annuum) and sunflower (Helianthus annuus). Although they exhibit different morphology, human-mediated selection has resulted in the enlargement of their reproductive organs, such as flowers, fruits and seeds ) measures the deviation of the F1 hybrid phenotype from a midpoint of the F0s. The additive effect (a = (X1−X2)/2) provides a quantitative estimate of the effect of substituting one allele, for example a wild allele for a mutant allele in a cultivated phenotype. Using a custom R function, we employed the normalized transcript abundance values and evaluated k for all the differentially expressed transcripts across 12 genetic crosses. We then sorted each transcript according to four discrete categories of gene action as follows: |k|< 0.25 = additive; |k|≥ 0.25 and |k|≤ 0.75 = partially dominant-recessive; |k|≥ 0.75 and |k|≤ 1.25 = dominant-recessive; |k|≥ 1.25 = transgressive. Beyond discrete categories of transcript abundance gene action, we analyzed the transcriptional landscapes employing probability distributions. This allowed us to compare the breath of allelic interactions in different contexts of selective pressures.We estimated the degree of dominance  at TFs and the number of genes they regulate (Out-degree). In a similar way, the number of TFs that regulate a single gene (In-degree) has been negatively be correlated with the ES. We thus employed a Pearson correlation analysis and a Loess non-linear regression to investigate the possible association between ES and both the Outdegree and the Indegree.k). We predicted that TFs with high Outdegree would tend to show additive gene action (|k|~ 0). We have no clear prediction for the inheritance of genes with high Indegree. We employed correlation and non-linear regression analyses to investigate the association between those network metrics and the degree of dominance (k).We were also interested in assessing the association between both Indegree and Outdegree to the degree of dominance (k), particularly for TFs showing partial or complete dominant-recessive gene action. We thus employed the absolute values of the Log2 fold change between the k values of TFs and their targets (|Log2(KREG/KTAR)|), to generate a normalized measurement of degree of dominance similarity. In addition to visualize the distributions of k similarity among the four categories of gene action of TFs, we explicitly compared the median similarity of TFs showing additive gene action to those showing non-additive gene action using a Wilcoxon rank sum test using the wilcox_test of the Rstatix R package [We reasoned that TFs and their targets would display similar degrees of dominance  across the array of genetic crosses. Color of dots indicate the significance of the enrichment; the size depicts the number of annotated genes to each GO. Figure S2. A sampling-based analysis revealed a uniform ~60X sequencing coverage for all the libraries used in the study. The per-transcript sequencing coverage  is shown as boxplots for the three genotypes of each species. The line within the boxes depicts the median, whereas the dots outside the box represent outlier values. Table S1. Summary of the per-species analysis of differential expression between parental and F1 hybrids. Table S2. Transcripts associated to domestication-related funcitons showing non-additive gene action. Table S3. Summary of the pair-wise differences between discrete categories of gene action of transcription factors and their target genes. Table S4. Details of the per-species RNA-seq libraries used in the study.


Biomolecules undergo continuous conformational motions, a subset of which are functionally relevant. Understanding, and ultimately controlling biomolecular function are predicated on the ability to map continuous conformational motions, and identify the functionally relevant conformational trajectories. For equilibrium and near-equilibrium processes, function proceeds along minimum-energy pathways on one or more energy landscapes, because higher-energy conformations are only weakly occupied. With the growing interest in identifying functional trajectories, the need for reliable mapping of energy landscapes has become paramount. In response, various data-analytical tools for determining structural variability are emerging. A key question concerns the veracity with which each data-analytical tool can extract functionally relevant conformational trajectories from a collection of single-particle cryo-EM snapshots. Using synthetic data as an independently known ground truth, we benchmark the ability of four leading algorithms to determine biomolecular energy landscapes and identify the functionally relevant conformational paths on these landscapes. Such benchmarking is essential for systematic progress toward atomic-level movies of continuous biomolecular function. Apart from disordered proteins, conformational continua span a spectrum of different energies. In thermal equilibrium, the probability of a conformational state being occupied is determined by the Boltzmann factor, which drops exponentially with increasing energy.Biomolecular machines have evolved to perform specific tasks through a concerted sequence of conformational motions. There is growing recognition that such motions involve continuous conformational changes, rather than jumps between a small number of discrete states4. In principle, an unlimited number of conformational paths connect a “start” conformation A to an “end” conformation B. However, most such paths include high-energy states, which are sparsely populated under biologically relevant conditions. Due to the exponential nature of the Boltzmann inverse relationship between energy and occupation probability5, lowest-energy conformational paths contribute maximally to function.Conformational motions of proteins can thus be represented as low-lying (and thus strongly occupied) pathways on one or more energy landscapes (EL)6, CryoSPARC 3DVA7, Manifold-EM8, and CryoDRGN VAE9, in faithfully extracting the energy landscape from snapshots. We benchmark the performance of each algorithmic approach in terms of the accuracy with which the correct energy landscape is recovered from the data.The growing recognition of the importance of energy landscapes for discerning function has spawned an increasing number of sophisticated algorithms capable of mapping continuous conformational motions. Using a synthetic dataset of cryo-EM snapshots with known ground truth energy landscape, we compare the performance of four leading algorithms, specifically Relion MultibodyTo date, no comparative benchmarking study of the strengths and weaknesses of different data-analytical approaches for analyzing continuous conformations has been reported. The lack of comparative benchmarks hampers the assessment of the usefulness and reliability of different algorithmic tools in extracting information from experimental data.In this paper, we use a synthetic dataset generated with an a priori known “ground-truth” energy landscape to benchmark, in silico, the above leading algorithms for conformational analysis of cryo-EM data. Specifically, we quantify each method’s ability to: (a) Recover the correct energy landscape from synthetic cryo-EM datasets; (b) Reveal the functionally important conformational degrees of freedom; and (c) Identify the functionally relevant conformational paths on these landscapes. Although the nature and number of potentially useful algorithms are currently evolving, the four selected approaches represent the state of the art in mapping continuous conformational motions from ensembles of single-particle cryo-EM snapshots.(i)Benchmark the performance of the four leading algorithms listed above in faithfully extracting conformational energy landscapes from synthetic cryo-EM snapshots, and(ii)Provide a well-characterized synthetic cryo-EM dataset suitable for comparative benchmarking, in order to facilitate the development of more effective data-analytical tools capable of identifying functionally relevant conformational landscapes and motions.The primary goals of this paper are thus twofold:The synthetic dataset of three million cryo-EM snapshots  stems from a ribosome-like object with two conformational degrees of freedom, with an underlying energy landscape of 12 energy minima of various depths arranged on a 3 × 4 grid  conformational coordinates were examined. We assess the performance of each algorithm in terms of its ability to classify snapshots correctly into the 12 ground truth energy minima. As outlined below, this approach allows us to quantify algorithmic fidelity in terms of well-known metrics from classification.10 defined asWe use “Recall”11 or simply Accuracy, allows us to assign a quantitative score to each algorithm’s ability to accurately extract the energy landscape underlying the synthetic data.  is simply an alternative representation of the conformational energy landscape of the ribosome-like object. Figure The occupation probability of a conformational state is determined by the energy of the state via the Boltzmann factor. An occupancy map .Compute the distance matrix (squared Euclidean) using the 12 centersUsing the distance of each particle from the corresponding center Fig. a, bin thThis procedure assigns particles to a label from 1 to 12.Finding an appropriate metric for extracting conformational landscapes from cryo-EM data is a challenging task, if only because the metric must quantify the distortion caused by the algorithmic approach. We tackle this problem by using the quantitative metrics Recall and Accuracy11 of the benchmarked method is given by:The AccuracyRecall ranges from 0 to 1 (as evident from the definition), where a value of 0 implies no snapshot is correctly assigned to that energy minima and a value of 1 when all snapshots belonging to the energy minima are correctly allocated. The accuracy of each algorithm is the average recall metric for assignment into all 12 minima.Supplementary Information.


The IMPAACT P1107 researchers presented the case details during the oral abstract session held at the 29th Conference on Retroviruses and Opportunistic Infections (CROI 2022). The study participant is a woman from New York (USA) who stopped antiretroviral therapy (ART) at 37 months post-transplant and has had no HIV detected for 14 months. The dual stem cell therapy also led to remission from leukemia that she developed in 2017.The IMPAACT P1107 is an observational study that aims to describe the outcomes in people living with HIV who undergo a transplantation with cord blood stem cells with a CCR5 genetic mutation for treatment of cancer, hematopoietic disease, or other underlying disease. This genetic mutation results in T cells without CCR5 co-receptors. As HIV needs to use these co-receptors to infect T cells, the rationale of the study is that chemotherapy given to people with cancers or other illness, followed by a transplant using stem cells that carry this CCR5 mutation, can change the immune system to make it genetically resistant to HIV.HIV remission, or cure, resulting from stem cell transplants had been previously reported in 2 cases. The first case, known as the Berlin patient (a man with acute myelogenous leukemia), was reported in 2009. He was submitted to a bone marrow stem cell transplant and experienced HIV remission for 12 years. He died of recurrent leukemia in September 2020. The second case, known as the London patient (a man with Hodgkin lymphoma), was reported more recently and has been in HIV remission for more than 30 months after a bone marrow stem cell transplant. This third case of HIV remission, now documented in a woman, suggests that a dual stem cell transplantation strategy could also be considered as an option to achieve HIV remission and cure for people living with HIV who require a stem cell transplant for other diseases.This case further supports the proof of concept for an HIV cure using stem cell transplants, but it is important to highlight that this approach is an invasive, complex and risky medical procedure and still not viewed as a feasible strategy to scale up to the millions of people living with HIV globally. Furthermore, as this CCR5 mutation is very rare , the chances of finding a suitable stem cell donor are very low.“Despite of feasibility challenges, this new HIV remission case is very exciting news and will continue to energize the HIV cure research agenda, reminding us of its potential to beat HIV,” says Dr Meg Doherty, Director of WHO’s Global HIV, Hepatitis and STI Programmes.More details about the caseThe HIV cure case described at the CROI 2022 involves a middle-aged woman of mixed-race ancestry who had developed high-risk acute myeloid leukemia while on ART, 4 years after a diagnosis of acute HIV infection. She achieved the leukemia remission after conventional leukemia chemotherapy and her HIV disease was also well controlled but with detectable virus. In 2017, she received a transplant of umbilical cord blood stem cells with a CCR5 mutation, supplemented with donor’s stem cells from the bone marrow of an adult relative. After receiving this dual stem cell transplant, she was engrafted with 100% cord blood cells at day 100 and had no detectable HIV. At 37 months post-transplant, the patient stopped ART. According to study team, no HIV was detected in the patient for 14 months except for a short transient detection of trace levels of HIV DNA in the patient’s blood cells at 14 weeks after stopping ART.About the dual stem cell transplant approachThe dual stem cell (or haplo-cord) transplant is a new transplant strategy that involves the transfusion of stem cells from an umbilical cord of neonates, complemented with stem cells from the bone marrow of an adult donor. This dual transplant process has been used in some individuals with high-risk cancers and requires less restrictive human leucocyte antigen (HLA) sample matching than adult- stem cells-only transplants, and it also makes the transplant procedure faster and safer. In this case, the cells of the umbilical cord had the CCR5 mutation and the adult stem cells also didn’t require an identical HLA matching, which is especially difficult to get for patients of African or mixed-race ancestry.https://www.who.int/news/item/24-03-2022-first-case-of-hiv-cure-in-a-woman-after-stem-cell-transplantation-reported-at-croi-2022Available from:


All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.The importance of air purifiers has increased in recent years, especially with the “coronavirus disease 2019” pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes  or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released  The disinfection of room air by suitable filter systems has become increasingly necessary particularly during the “coronavirus disease 2019” (COVID-19) pandemic . Based o10 step) within half an hour when used as intended . Tw. Tw18]. After adding a nuclease to a particular sample, three result outputs can be delivered. First, samples containing only intact virus particles give the same result, regardless of whether pretreatment with the nuclease was carried out or not . The secThe efficacy of the universal nuclease as well as the viability dye PMA was first tested with FCoV RNA in triplicate. The universal nuclease was able to degrade the entire RNA . In contThe effect of the universal nuclease on intact virus particles represented by a dilution series of a cell culture supernatant was verified in duplicate. In both RT-RAA and real-time RT-PCR, the dilution series with pretreatment shows almost the same results as the dilution series without pretreatment , which mThe dilution series of cell culture supernatant  was enriched with FCoV RNA. The mixture was pretreated with universal nuclease. In both RT-RAA and real-time RT-PCR, only the RNA was amplified from the intact virus particle as the free RNA was degraded when pretreated with the universal nuclease . This indicates that the enzyme mixture works with various amounts of virus particles.The first UV-C lamp was able to inactivate the viruses with 100% efficacy. The samples collected in front of the air purifier (collection point #1) resulted in a signal in both RT-RAA  as well 50 in cell culture.Air purifiers play an important role in prevention of airborne viral diseases and must be tested according to official standards before commercial use. The gold standard method is determining the infectivity using cell culture-based approach, which is time-consuming , 19. In There are several methods to evaluate air purifiers, including animal experiment  as well In the present study, a comparison was first made between the viability dye PMA and the universal nuclease. The PMA revealed a poor performance in our set up. PMA proved to be a promising approach for distinguishing between viable and non-viable bacteria  and has The universal nuclease resulted in complete degradation of free RNA in our study. The search on PubMed revealed few results on the use of nuclease-like benzonase in viability assays of microorganisms. Two studies successfully applied enzymes to degrade free nucleic acids , 51. Mon2) are able to destroy the NA as well as the capsid [The samples collected behind the air purifier of UV-C were completely inactivated. UV irradiation can damage the NA, while the protein coat remains intact and protects the NA . This woe capsid .In previous studies on the efficacy of air purifiers, a specific pathogen is often aerosolized and tested for infectivity at the end of the test procedure by culturing bacteria or viruses , 55, 56.


With the surge of the high-throughput sequencing technologies, many genetic variants have been identified in the past decade. The vast majority of these variants are defined as variants of uncertain significance (VUS), as their significance to the function or health of an organism is not known. It is urgently needed to develop intelligent models for the clinical interpretation of VUS. State-of-the-art artificial intelligence (AI)-based variant effect predictors only learn features from primary amino acid sequences, leaving out information about the most important three-dimensional structure that is more related to its function.Methods: We proposed a deep convolutional neural network model named variant effect recognition network for BRCA1 (vERnet-B) to recognize the clinical pathogenicity of missense single-nucleotide variants in the BRCT domain of BRCA1. vERnet-B learned features associated with the pathogenicity from the tertiary protein structures of variants predicted by AlphaFold2.Results: After performing a series of validation and analyses on vERnet-B, we discovered that it exhibited significant advances over previous works. Recognizing the phenotypic consequences of VUS is one of the most daunting challenges in genetic informatics; however, we achieved 85% accuracy in recognizing disease BRCA1 variants with an ideal balance of false-positive and true-positive detection rates. vERnet-B correctly recognized the pathogenicity of variant A1708E, which was poorly predicted by AlphaFold2 as previously described. The vERnet-B web server is freely available from URL: http://ai-lab.bjrz.org.cn/vERnet.Conclusions: We applied protein tertiary structures to successfully recognize the pathogenic missense SNVs, which were difficult to be addressed by classical approaches based on sequences. Our work demonstrated that AlphaFold2-predicted structures were expected to be used for rich feature learning and revealed unique insights into the clinical interpretation of VUS in disease-related genes, using vERnet-B as a discovery tool. AlphaFold2 overcame the enormous economic and time demands in determining protein structures and enabled the efficient obtaining of more tertiary protein structures. Compared with the widespread technique, homology modeling (HM) Identifying the pathogenicity of human disease-associated gene variants, especially missense single-nucleotide variants (SNVs), is crucial for clinical decisions. For example, deleterious genetic mutations in BRCA1, a tumor suppressor gene, can impair protein function and raise the risk of multiple cancers Therefore, we developed a learning model for inferring protein function from their tertiary structures, which facilitates the functional classification of BRCA1 variants relying on their tertiary structures with deep learning. We collected missense SNVs with known pathogenicity in the BRCT domain of BRCA1 from ClinVar Our method aimed to learn the pathogenic propensity of human missense SNVs in BRCA1 from the protein tertiary structures  spectroscopy, and cryo-electron microscopy (cryo-EM) Since missense SNVs only cause the substitution of one amino acid, they usually have little effect on the coiling and folding of the protein structures, resulting in a mean value of average Cα r.m.s.d. of only 0.25Å when comparing all variants to WT  for enhancing the pathogenicity recognition capacity. MutPred2 was also developed by machine learning with an output of the pathogenicity score ranging from 0 and 1, in which the higher score indicates the greater possibility of pathogenicity We further investigated the effectiveness of various features in ANNs for our model. There are three features with a single type of interaction, including hydrogen bonds (hbond), overlaps (ovl), and interatomic contacts (cnt). The weighted combination of these single features was added into the networks as a new feature, generic residue interaction (combi). Each feature was individually used to train a model, which was also evaluated by the testing dataset with 484 variants. As shown in Figure vERnet-B is unable to be directly applied to other genes for pathogenicity recognition. In order to precisely identify the effect of small structure changes caused by one amino acid substitution, our method required that an individual recognition model should be trained for each protein. For example, vERnet-B failed in pathogenicity recognition for the BRCT domain of BARD1. BARD1 has homology BRCT domain of BRCA1, and the stable interaction between BRCA1 and BARD1 is essential for BRCA1 as a tumor suppression To validate our method and demonstrate its value as a discovery tool of variant interpretation, we applied this schema to the RING domain of BRCA1, which was a fragment containing 104 amino acids independent of the BRCT. We collected 521 missense SNVs in RING from the aforementioned platforms, in which the 279 variants were used to train a model for recognizing the pathogenicity of RING variants see . This mohttp://kinemage.biochem.duke.edu) https://rinalyzer.de/rinerator.php) Construction of AANs for protein variants in the current datasets (1143 variants) was performed to train a deep-learning model vERnet-B or evaluate its performance. As some successful practices of using AANs to infer protein functions have been achieved in previous studies https://www.ncbi.nlm.nih.gov/clinvar) https://brcaexchange.org) https://sge.gs.washington.edu/BRCA1) Six hundred and fifty-nine BRCA1 variants were selected and downloaded from three platforms, including ClinVar (The architecture of the learning model used to implement the base classifiers of vERnet-B was ResNet18 One of the popular methods in dealing with class-imbalance problems is under-sampling. The traditional under-sampling methods only used a subset of the majority class This work succeeded in the recognition of the pathogenicity of missense SNVs in BRCA1. vERnet-B considerably outperformed other computational methods in terms of accurate and unbiased recognition of variant pathogenicity, and combining vERnet-B with other evidence further enhanced the model performance. vERnet-B has the potential to facilitate the clinical application of genetic information by interpreting VUS. For example, determining the appropriate classification of VUS can aid genetic counseling The fact that the majority of variants in ClinVar still had uncertain or conflicting significance illustrated the challenges posed by VUS. The traditional approaches for interpreting VUS were experimental assessments The potential problem caused by the inconsistent interpretation of several variants in our raw dataset was a caveat of the current research. Although we had deleted some of these ambiguous data, some unfaithful samples with single evidence might still be included. We have carried out a simple solution, cross-training, to eliminate the influence of such unfaithful samples. We pre-trained a CNN model using some independent samples to recognize the training dataset. Two samples that were incorrectly recognized with extreme scores, which indicated they were difficult samples or misclassified samples, were removed from the training dataset. Optimized data screening methods are expected in future studies.The major limitation of this method was the inevitable conflict between precision and generalization ability. The current method required training an individual recognition model for each protein. While the vERnet-B cannot be directly generalized to other genes, we had a successful application on RING domain of BRCA1 by training an individual model for it. It is conceivable that further investigation on this method will make it more useful by getting a better balance between precision and generalization.AlphaFold2 provides an optimal static structure, while the state of compound binding may cause conformational changes in proteins. However, although the molecular dynamics simulation is theoretically appealing, this approach is highly challenging for even moderate-sized proteins due to the complexity and context dependency of protein stability In conclusion, we proved that the relationship between AlphaFold2-predicted protein tertiary structures and the pathogenicity of missense SNVs is learnable. The construction of AANs is a critical step for applying protein tertiary structures to deep learning. This method paved the way for generating variant effect learning models with even higher accuracy in the future. vERnet-B will spur clinical genetic information research and enrich the toolbox of design and implementation of precision treatment.Supplementary figure and tables.Click here for additional data file.


Psychotic disorders often cause a drastic change in the life situation of the affected individual. Personality is an aspect that can affect the symptoms and social function in psychotic disorders.No study has examined stability of personality traits exceeding five years in patients with schizophrenia. The aim of this study was to investigate the stability of personality traits over a 13-year period among patients with psychotic disorder and healthy individuals and to evaluate case-control differences.At three occasions during a 13-year period patients with psychotic disorders (n=28) and non-psychotic individuals (n=57) completed Swedish universities Scales of Personality (SSP). For all the individuals within- and between-subject analyses were performed at three occasions for all 13 subscales and the three overall factors of SSP. Correlations, means and SDs were calculated.Tests of within-subject correlations showed differences in two subscales: Lack of Assertiveness, which were influenced by age and Physical Trait Aggression, where patients ratings were stable, whereas controls rated themselves less aggressive at higher age. Between-subjects correlations showed differences regarding any of the parameters diagnosis, time, age, gender or age x gender in nine of the 13 subscales as well as in factor Neuroticism.Long term follow-up showed a generally high stability of personality traits measured with SSP, especially among patients. Between-subject analyses over the 13 years showed that patients differed compared to controls for the SSP factor Neuroticism as well as the subscale Detachment, which is in accordance with previous studies.


Spatial navigation is an intricate ability, requiring multisensory and motor integration, that is particularly impacted in aging. The age-related decline in navigational capabilities is known to be associated with changes in brain regions such as the frontal, temporal, and cerebellar cortices. Age-related cerebellar differences in spatial navigation have generally been ascribed to motor impairments, omitting the central role of this structure in several cognitive processes. In the present voxel-based morphometric study, we investigated gray matter volume loss in older adults across cognitive and motor subregions of the cerebellum. Specifically, we hypothesized that age-related gray matter differences would occur mainly in cerebellar regions involved in cognitive processing. Our results showed a significant age-related atrophy in the left neocerebellum of healthy older adults that includes Crus I and lobule VI. The latter are important nodes in the network that subtends cognitive abilities such as object recognition and spatial cognition. This exploratory work sets the ground for future research to investigate the extent of the neocerebellum’s contribution to spatial navigation deficits in aging. Navigating in space constitutes a key factor to maintain older adults’ autonomy and mobility and to prevent the occurrence of age-related disorders. Healthy older adults show impairments in spatial navigation ability that include disorientation in unfamiliar environments and difficulties in taking detours to avoid obstacles . This deIn the healthy aging literature, a handful of studies have reported age-related changes in cerebellar volume or activity associated with worse navigational performance . HoweverThe present study is a re-analysis of anatomical data from 25 younger  and 17 older participants . All participants completed a battery of neuropsychological tests including the Mini-Mental State Examination , the 3DData were acquired using a 3-Tesla Siemens MAGNETOM Skyra whole-body MRI scanner  equipped with a 64-channel head coil. T1-weighted high-resolution three-dimensional images were obtained using an MPRAGE sequence .http://www.fil.ion.ucl.ac.uk/spm/) implemented in MATLAB 9.4. Each individual scan was manually reoriented to set the origin  on the anterior commissure and thus isolate segmented maps of the cerebellum. Manual quality control was performed to correct potential segmentation errors such as GM located outside of the cerebellum. Images were then normalized using the diffeomorphic anatomical registration through exponentiated lie algebra algorithm (DARTEL) [t-test to compare GM volume between young and older adults. Second, we performed a multiple regression analysis to investigate the associations between navigation time and error rate and GM volume of cerebellar subregions. For all analyses, brain size and gender were included as covariates of no interest. The statistical threshold was set at p < 0.05 family-wise-error (FWE) corrected at cluster-level with a minimum cluster extent of 20 voxels.Data processing was performed using the cerebellar-specific toolbox SUIT –19 and t(DARTEL) , 22, modp < 0.001 FWE cluster-level) in the left neocerebellum of healthy older participants . No other significant association between neuropsychological scores and GM volume was found. A supplementary whole-brain analysis showed that reduced GM volume in the right entorhinal cortex only was related to an increase in navigation time  found in older adults could be interpreted in light of previous fMRI results from . RamanoëGiving support to this hypothesis, our results appear to overlap with the functional task-related cerebellar atlas for the use of a basic spatial map by King and colleagues . We need to highlight that the present study failed to identify a significant association between navigational performance and GM volume in the cerebellum. These results could be partially explained by the limited sample size but also by the relative simplicity of the navigation paradigm involving a two-choice task based on a small number of simple objects. However, we did show significant association between GM volume and scores on the PPT in lobule VI. This result and the absence of other significant correlations, notably with tests assessing specific memory processes, could be also explained by the capacity of the PPT to reflect multiple facets of cognition , 29. PreThese preliminary findings warrant further research with a larger sample size and ecological navigation paradigms that could disentangle the cognitive and motor aspects of spatial cognition. Despite several limitations, our results reveal age-related atrophy in Crus I and Lobule VI, regions which are specifically implicated in the cognitive aspects of spatial navigation . We emph


Cupressus sempervirens var. horizontalis) in the Zagros Mountains, Iran. Furthermore, we separately measured earlywood width (EWW) and latewood width (LWW) of Persian oak (Quercus brantii Lindl.) to examine if intra-annual resolution of tree-ring parameters of Q. brantii tree rings can be used as high-resolution paleoclimate proxies. Climate-growth relationships revealed that mean monthly maximum temperatures (Tmax) are a dominant factor determining radial tree growth and negatively affect both oak and cypress in the Zagros Mountains. Accordingly, we reconstructed two different seasonal windows of past Tmax variability, namely, January–March and June–August over the periods 1860–2015 and 1560–2015, respectively. Regime shift detection identified twelve warm and nine cold significant regime shifts in our summer Tmax reconstructions. The longest hot summer period occurred in the twentieth century, and two warm regime shifts occurred in 1999 and 2008. The highest values of the warm summer regime shift index occurred in 2008, which coincided with fungal pathogen attacks and insect outbreak of the oak leaf roller moth (Tortrix viridana L.) in the Zagros oak woodlands. Interestingly, we found common warm and cold periods in historic climate variability between the summer and winter Tmax reconstructions. Warm and cold regime shifts occurred simultaneously from 1955 to 2015, and significant regional warm summer and winter regime shifts have occurred between 2008 and 2015. The winter and summer Tmax reconstructions show high spatial correlations with large areas in West Asia, North Africa, and the eastern Mediterranean region. Our results strengthen initial studies on past climate variability in Iran and contribute to an enhanced understanding of past temperature variability in West Asia.Abrupt changes in temperature have especially strong impacts on fragile ecosystems located in semi-arid regions. In this study, we analyzed tree-ring widths (TRW) of Mediterranean cypress (The online version contains supplementary material available at 10.1007/s00484-022-02380-5. Climate change and its negative impacts on local communities and regional ecosystems are among the major challenges of our time IPCC . IncreasLocated in the transition zone between the westerly dominated temperate and subtropical zones, Iran is highly vulnerable to the negative impacts of temperature extremes. Based on Iran’s Metrological Organization (IRIMO), the country’s mean temperature has increased by 2 °C over the last decades (1968–2020), considerably higher than the global average. Instrumental climate data show rapidly increasing trends of both, maximum and minimum temperatures in Iran  to reconstruct past seasonal variability of Tmax using annually and intra-annually resolved tree-ring series of C. sempervirens and Q. brantii; (3) to identify the forcing mechanisms responsible for the variations in seasonal Tmax in the semi-arid region of Iran, including climate regime shifts; and (4) to investigate related influence of climate change on the regional oak decline and insect outbreaks in oak woodlands in the Zagros Mountains.The main objectives of this study are (1) to investigate how Quercus brantii Lindl.) as the dominant tree species. Q. brantii is a native ring-porous species in the Zagros Mountains growing at elevations between 700 and 2300 m a.s.l. At higher elevations, oak forests are replaced by a more open forest type composed of more drought-tolerant tree species. In the southern Zagros Mountains, these forest steppes are locally mixed with Cupressus sempervirens var. horizontalis (Mill.), an evergreen coniferous species growing in small colonies in some valleys, between 1220 and 2000 m a.s.l. in Tang-Soulak site.The northwest to southeast orientated Zagros Mountain range in Iran is a part of the Fertile Crescent. The mountainous region serves as a water tower for the surrounding arid and semi-arid basins and has significant impacts on atmospheric circulation through physical and thermal dynamic effects over the Middle East  and Tang-Soulak  are located in the southern parts of the Zagros Mountains  with a precision of 0.01 mm coupled to the analytical software TSAP-Win Rinn . Ring-wiExpressed population signal  and minimum (Tmin) temperatures and monthly precipitation (P) of the Yasuj meteorological station  were used to calculate climate-growth relationships. As analytical time windows, we investigated Tmax and Tmin monthly data from January of the previous growing year until September of the current year, as well as different seasonal windows. Monthly precipitation from October of the previous growing year to May of the current year was investigated since the region only receives precipitation during this period.r =  − 0.68, p < 0.01) and TS-TRW  chronologies and January–March Tmax (hereafter DB-JFM) and June–August Tmax (hereafter TS-JJA), seasonal Tmax were reconstructed using linear regression models. We tested the autocorrelation of climate data prior to developing the regression models. Due to the short length of the available instrumental climate data series (1987–2015), the leave-one-out cross-validation method  to assess the regional significance and the representativeness of the seasonal Tmax reconstructions. We also conducted climate regime shift analysis using the Shift Detection Program version 3.2 (www. beringclimate.noaa.gov/regimes/) to identify abrupt regime shifts in the summer and winter Tmax reconstructions and to characterize the patterns of Tmax fluctuations in the seasonal reconstructions. The Shift Detection Program is based on a sequential Student’s t-test calculated for a 10-year window moving over the mean values of the reconstructions , the Pacific Decadal Oscillation  to investigate the influence of atmospheric circulation patterns on seasonal Tmax in the Zagros Mountains. NAO is a large-scale atmospheric pressure seesaw over the North Atlantic Ocean which is based on the surface sea-level pressure difference between the subtropical high and the subpolar low and has a strong effect on winter weather in Europe, Greenland, and northern Asia. SNAO represents the strength of a spatial pattern of pressure variability in the summer (July–August) over the northern North Atlantic Ocean. AO is a shifting of atmospheric pressure between the Arctic and the mid-latitudes of the North Pacific and North Atlantic, and positive and negative AO phases can affect temperatures in mid-latitudes. AMO is a periodically variation in the sea surface temperature (SST) of the North Atlantic Ocean that may last for several decades, and it is based on the average anomalies of SST in the North Atlantic Ocean with cool and warm phases. PDO is a long-term fluctuation of SST over the Northeastern Pacific Ocean, and its positive and negative phases can influence the atmospheric circulation patterns.We calculated correlations between the reconstructed DB-JFM and TS-JJA and the North Atlantic Oscillation  was reached after 1860 and 1560 C.E. for DB-EWW (6 trees) and TS-TRW (8 trees) chronologies, respectively. DB-EWW chronology shows similarities with TS-TRW chronology over the common period  showed stronger mean inter-series correlation than TS-TRW (r = 0.40).DB-EWW 1805–2015) and TS-TRW chronologies (1450–2015) from the southern Zagros Mountains and their statistical characteristics are shown in Fig. 015 and TQ. brantii and C. sempervirens  with DB-EWW during the current year (January to March). Tmax showed the highest negative correlations with the TS-TRW chronology during the previous year March and June and the current year June. It should be mentioned that Tmax showed strong negative correlations with the TS-TWR chronology during the previous and current year June. Correlations between seasonal Tmax and the TS-TRW chronology showed a strong significant negative correlation during June to August, while DB-EWW shows strong significant negative correlations with seasonal Tmax during January to March.Evaluation of climate growth relationships revealed that Tmax from previous January to current year September, which has negative effects on the growth of ens Fig. a, showedTmin has negative relationships with DB-EWW chronology during the current growth year Fig. , whereasThe linear regression models for the reconstruction of seasonal Tmax in DB-JFM and TS-JJA fulfill all statistical verification tests Table  and confr = 0.68, p < 0.01) and TS-JJA  reconstructions and instrumental climate data revealed high correlation between the time series over their common period 1987–2015 , and most of the warm (42) and cold (45) years occurred simultaneously. The increasing trends of winter and summer Tmax started around 1965 after a relatively long cold period, though it should be mentioned that recent Tmax values in winter do not exceed previous maxima. The increasing trend of the reconstructed summer Tmax from 1980 to 2015 is stronger than that of winter Tmax.DB-JFM and TS-JJA reconstructions cover 156 1860–2015) and 456 (1560–2015) years, respectively  and one cold (1982–1998) periods in DB-JFM and TS-JJA reconstructions during the common period 1860–2015 Fig. , b. DB-JSpatial correlations between the reconstructed DB-JFM and regional gridded Tmax  and AO (1950–2015) and DB-JFM in the Zagros Mountains. Also, significant negative correlations were found between SNAO and PDO and the reconstructed TS-JJA. AMO showed positive relationships with the reconstructed DB-JFM and TS-JJA Fig. , b.Q. brantii make this xerophilous oak species very suitable for seasonal climate reconstructions in the Zagros Mountains  and show similar patterns during their common period (1860–2015). However, contrasting patterns between the two reconstructions occurred during 1900–1915 and 1930–1950 and 1984–1990. The TS-JJA reconstruction indicates that generally warmer conditions and the longest warm summer period occurred in the twentieth century. This warming trend was also reported by Köse et al. , which is a common early season defoliator of oaks . The simultaneous occurrence of fungal pathogen attacks and outbreak of oak leaf roller moth (Tortrix viridana L.) with the warm regime shifts and rapid increasing trends in summer and winter Tmax highlight the strong impacts of the ongoing climate change on the oak woodlands and fragile ecosystems in the Zagros Mountains.The highest regime shift in 1971 and 2008 and consecutive warm regime shifts at the end of twentieth century and at beginning of twenty-first century confirm the intensity of the Tmax variability and occurrence of record-breaking temperatures during the last decades in the Zagros Mountains. During the twentieth century shifts between the summer and winter reconstructions occurred simultaneously, confirming the effects of the global warming trend on temperatures in all seasons in the mountainous region of southwest Iran Fig. , b. An iAs a dominant mode of the Atlantic’s sector climate variability, NAO controls heat and moisture fluxes from the Atlantic Ocean into the Mediterranean region Türkes  and chanQ. brantii) and coniferous (C. sempervirens) trees in the semi-arid region. Abrupt increases in winter Tmax (January–March), which strongly affect the earlywood part of Q. brantii tree rings, played an important role in stimulating outbreaks of invasive insects and fungal pathogen attacks in the Zagros oak woodlands during the last years (2008–2015), highlighting the vulnerability of the xerophilous oak species to ecological effects of global warming.In this study, we presented two new reconstructions of winter and summer Tmax based on DB-EWW (1860–2015) and TS–TRW (1560–2015) in the Zagros Mountains, Iran. Tmax in the winter and summer have strong negative impacts on the deciduous Below is the link to the electronic supplementary material.