There is a significant negative correlation between emergency escape capability and LF_increase_rate (p < 0.05), and a remarkably significant negative correlation between emergency escape capability and LF/HF_increase_rate (p < 0.01); the greater the increase rate of LF or LF/HF, the smaller the emergency escape capability, with excessive stress probably not being conducive to emergency escape. There is a very significant negative correlation between an individual’s emergency escape capability and the degree of familiarity with the Zijing Mountain subway station (p < 0.01). The findings provide references and suggestions on the emergency management and emergency evacuation for government and subway departments.Studying an individual’s emergency escape capability and its influencing factors is of great practical significance for evacuation and escape in subway emergencies. Taking Zhengzhou Zijing Mountain Subway station as the prototype, and using VR technology, a virtual subway fire escape scene was built. Combined with the total escape time, the total contact time with fire, and the total contact time with smoke, it proposed a calculation formula on emergency escape capability. A total of 34 participants with equal gender distribution were recruited to carry out the virtual subway fire escape experiment, and participants’ physiological data  were real-time recorded by ErgoLAB V3.0 throughout the whole experiment. The emergency escape capability of each participant was evaluated quantitatively, and the related influencing factors were analyzed. The results show that for the age ranges (19–22 years old) in the experiment, the emergency escape capability of women is significantly lower than that of men ( As an important part of a modern urban public transportation system, the subway is an effective tool to relieve traffic congestion. However, subways are a limited underground space with crowded occupants, who are characterized by high density, high mobility, and complex composition. Once a fire occurs, a large number of casualties and economic losses can be caused, such as the Daegu subway fire in South Korea in 2003 , which cUsually, in an evacuation model, data about the evacuee’s pre-reaction time, movement speed, escape behavior, exit choice, individual characteristics, the influence of obstacles in the moving path on them, and the escape time are often considered to be collected . The comDue to the “3I”  features, virtual reality (VR) technology has been widely applied in different fields, such as military ,14, mediFrom the view of personal characteristics, evacuation can be affected by age, gender, physical condition, physiological and psychological quality, personality type, etc. Zijlstra et al.  simulatePrevious studies have shown that certain physiological indicators of individuals will change regularly under stress in case of emergency. In the virtual experimental study of Tucker et al. , Meng etEvacuation could also be influenced by an individuals’ personality traits and emotions . Alexia A total of 34 undergraduates from Zhengzhou University, 17 men and 17 women, mean age = 20.32 ± 0.88 years, ranging from 19 to 22 years old, were recruited to carry out the experiment. All participants had normal or corrected-to-normal vision, without any color vision deficiencies . In addiAll software and hardware used in the experiment are shown in https://lbs.amap.com/api/javascript-api/example/map/map-english/ (accessed on 23 September 2021)), which is the largest transfer subway station of Line 1 and Line 2 in Zhengzhou, China, with a high passenger flow every day, as the prototype to build a VR subway fire escape scene using SketchUp and Unity3D, with 1:1 scale of the real station [Here, we chose Zhengzhou Zijing Mountain subway station Settings of fire, smoke, illumination, and brightnessThe fire and smoke in the virtual scene were set by the particle system of Unity3D. According to the collision mechanism, the system would automatically count the contact time of the virtual character with fire or smoke. Vilar et al. ,49 found(2)Settings of the starting point and ending point of the escapeThe whole fire escape process includes two stages: the train escape stage and the station escape stage. The whole escape begins at the fire occurring on the train of Line 2 in Level-4 and ends with the participant’s arrival at the ground exits of the subway station. In the train escape stage, when the fire first occurs on the moving train of Line 2, participants need to perform a series of actions to get off the train, such as reporting the fire to the train driver through the emergency communicator, trying to put out the fire with the fire extinguisher, and opening the train door with the emergency door opener after the train arrives at the subway station. When participants get off the train and enter the subway station, they will start their station escape stage and need to continue their station escape until they arrive at the ground exits of the subway station.(3)Settings of itemsFire extinguishers, towels, and bottled water are set in the virtual scene. Participants can use fire extinguishers to put out the fire on their escape paths, or pick up the towels and bottled water to resist fire and smoke, all of which can reduce their total contact time with the fire and smoke, and consequently change their emergency escape capability score. Participants can also pass through the fire or smoke directly during the escape, but their contact time with fire or smoke will increase, and thus change their final emergency escape capability score.(4)Settings of the moving speed of the virtual charactersIn the real emergency escape, the moving speed of people is generally 0.1–1.98 m/s . In the (5)Setting of the maximum escape timeGamberini et al.  set a ma(6)Setting of the virtual teaching and practice sceneIn order to make participants familiar with the virtual experiment operation before the formal virtual fire escape experiment, we first developed a virtual teaching and practice scene, which can teach and practice participants’ movement, the use of fire extinguishers, and the use of the emergency door opener, etc. in the virtual environment see .(1)t is the score deduction related to the total escape time, which is the penalty points for total escape time exceeding 300 s. Df is the score deduction related to the fire contact, which is the penalty points for contacting fire. Ds is the score deduction related to the smoke contact, which is the penalty points for contacting smoke.The formula of emergency escape capabilityIn order to quantify participants’ emergency escape capability in a subway fire escape, according to their total escape time, their total contact time with fire, and their total contact time with smoke in the whole virtual subway fire escape, the following emergency escape capability formula was proposed:t, Df, Ds as 40, 20, and 40% respectively.In Equation (1), a point deduction system is adopted. The full score of the emergency escape capability is set to 100 points. According to the statistics, most of the deaths in fires are caused by smoke rather than open flames , also, c(2)t—Formula of penalty points for escape overtimet is the total escape time. Through the preliminary experiment, participants’ average total escape time is 300 s more or less, so here we set 300 s as the baseline of escape time, which is more strict because it is shorter than the 6 min of safety evacuation time that is stipulated in Code for Design of Metro (GB 50157-2013), Standard for Fire Protection Design of Metro (GB51298-2018), Code for Safety Evacuation of Metro (GB/T 33668-2017), and NFPA 130- 2017 (Standard for Fixed Guideway Transit and Passenger Rail Systems). The formula of Dt < 300 s, Dt is negative, then force Dt = 0. Namely, when Tt ≤ 300 s, then Dt = 0, which demonstrates that this participant’s total escape time is shorter, and there is no penalty points for escape overtime for him. When Tt = 600 s , Dt = 40. Namely, when 300 s < Tt ≤ 10 min, there will be penalty points for escape overtime for the participant, and the range of points penalty for overtime is 0 < Dt ≤ 40. 40% is the proportion of escape overtime deduction.In Equation (2), when T(3)f—Formula of penalty points for contacting firef is the total contact time with fire. 10 s is the maximum time set for the virtual character that can contact with fire in the system. If Tf > 10 s, the escape will automatically end. 20% is the proportion of points deducted for contacting fire.The formula of D(4)s—Formula of penalty points for contacting smoke.s is the total contact time with smoke; 20 s is the maximum time set for the virtual character that can be in contact with smoke in the system. If Ts > 20 s, the escape will automatically end; 40% is the proportion of points deducted for contacting smoke.The formula of DIn Equations (3) and (4), 10 s is the maximum time of contact with fire and 20 s is the maximum time of contact with smoke, and are relevant to the specific amount setting of fire and smoke in our virtual subway fire escape scene. Due to people being more likely to come into contact with smoke than with fire in a fire condition, the amount of fire set in our virtual subway fire escape scene was also less than the amount of smoke. In addition, considering it would be hard for people to stand too long in the high heat of fire and smoke, according to the preliminary experiment, participants’ total contact time with fire and smoke were both less than 10 s and 20 s, respectively, so here we set the maximum time of contacting with fire is 10 s, and the maximum time of contacting with smoke is 20 s. However, this value should be determined from numerical simulation, which is also the focus of our future study.After each participant finished his or her escape, the virtual scene will automatically jump to the results interface See . Before the formal experiment, all of the participants had to sign an informed consent, fill in their basic personal information see , such asFormal experiment phase: After the virtual practice, participants wore a PPG and EDA sensor and stayed calm for 2 min see a. Then, https://pan.baidu.com/s/1cAbFGt23qqtvdChkyzyN2g (accessed on 23 September 2021); Code: 7872). The data was analyzed by SPSS 22.0.Data were collected and initially processed by ErgoLAB  are plotted in c, and the red line represents all the participants’ average Sc.Thirty-four participants’ score of emergency escape capability , and the mean of Sc in women (75.37 ± 11.93) was significantly lower than that in men (83.79 ± 10.03). This reflects that within the age group (19–22 years old), men’s emergency escape capability was better than women’s. Gender is a factor affecting an individual’s emergency escape, which verifies Hypotheses 1 and is also consistent with the existing research [From the tests of normality and homogeneity of variance test in SPSS, the Snder see . From Taresearch .c in the four kinds of DISC personality types (p > 0.05).From the one-way ANOVA results in SPSS see , there wc in compliance personality type (83.09 ± 9.02) is the highest, and the mean of Sc in influence personality type (77.43 ± 9.42) is the lowest, which verifies Hypotheses 2. The reason may be that under the emergency stress, people with influence type are fickle and easily influenced by the surrounding environment, and have no their own opinion on the escape direction, while people with compliance type are more prudent at obtaining evacuation guiding information and can make correct decisions. This provides thoughts for targeted training on different personality types of occupants in the future.Some studies have shown that people with a dominance personality type usually make quick decisions and often show high initiative and vitality; people with an influence personality type are often fickle and curious; people with a steadiness personality type are patient, loyal, and good at creating a stable and harmonious atmosphere, and their biggest motivation is the sense of safety; people with a compliance personality type are careful and prudent, and can think ahead and prevent problems . AccordiThe SC and EDA data were processed by Gaussian filtering and low-pass filtering  in ErgoLAB V3.0. The HRV data were processed by Wavelet denoising, high-pass filtering, low-pass filtering, and band-hinder filtering  in ErgoLAB V3.0.Previous relevant studies have shown that when people are under stress, their physiological indicators will change. Usually, their SC will increase ,10. Meanp < 0.01, both d (effect size) > 0.8); SDNN, RMSSD, and LF/HF all decreased without significance ; LF, HF both increased without significance . These indicated that participants were under stress during their virtual fire escape. This was also verified by the post-questionnaire, in which more than half of the participants felt nervous in the virtual fire escape. From the tests of normality and homogeneity of variance test in SPSS, the physiological data during virtual fire escape and baseline period all obeyed normal distribution and homogeneity of variance, so the paired-sample T-test was used to determine the changes of physiological data during virtual fire escape compared to the baseline period. From the paired-sample T-test results see , during c, Mean_SC_increase_rate, Mean_HR_increase_rate, SDNN_increase_rate, RMSSD_increase_rate, HF_increase_rate all obeyed normal distribution, so the Pearson correlation test was used to determine the relationship between Sc and each physiological indicator. From the Pearson correlation results ; while there is a significant negative correlation between Sc and LF_increase_rate (p < 0.05), and a remarkably significant negative correlation between Sc and LF/HF_increase_rate (p < 0.01). This indicates that the greater the increase rate of LF and LF/HF, the smaller the Sc, while an increase of LF and LF/HF represents the person being in a stressful state, which may reflect that excessive stress is not conducive to emergency escape (which verifies Hypotheses 3). Therefore, during the whole emergency evacuation process, subway management departments should take various emergency management and emergency evacuation measures to maintain occupants’ emotional stability and avoid their excessive stress; for example, designing and setting effective dynamic flickering emergency evacuation guidance signs, ensuring power supply for emergency lighting, playing subway staffs’ roles as much as possible to guide occupants’ emergency evacuation, providing helpful evacuation information through broadcast, using laser-guided emergency evacuation technology and systems, etc.From the Tests of Normality in SPSS, the Sults see , there wc and other influencing factors. From the Spearman correlation results , which indicated that the experimental design in this paper eliminates the influence of certain irrelevant variables on the experimental results well. Furthermore, from c and difficulty in finding the escape exits (p > 0.05), while there was a very significant negative correlation between Sc and the degree of familiarity with the Zijing Mountain subway station (p < 0.01), which indicates that the more familiar with the subway station, the higher the Sc was. This provides a way to improve an individual’s emergency escape capability by becoming familiar with the station consciously, including familiarity with various escape routes and emergency exits after entering the subway station, etc.According to the post-questionnaire, 73.5% of the participants thought the virtual fire escape scenes were of a high degree of immersion; 73.5% of the participants had computer game experience; 32.4% of the participants had VR experience, which was mainly VR games or VR movies; 97.1% of the participants thought the operation in the virtual scene was general or not difficult or not difficult at all; 64.7% of the participants thought it was not difficult or not difficult at all to find the escape exits; 70.6% of the participants were unfamiliar or completely unfamiliar with the Zijing Mountain subway station. Here, a Spearman correlation test was used to determine the relationship between Sults see , the imm(1)Virtual sceneThe fire and smoke in the virtual scene were not numerically simulated, the pattern of which may not conform to the real condition and limit the immersion. In future, fire and smoke will be numerically simulated to provide a more realistic scene visually. In addition, there were no NPCs set in the virtual escape scene without considering the characters’ social force impact. More NPCs will be added to the virtual scene in future studies.(2)The formula of emergency escape capabilityIn the real fire environment, there are various complex factors affecting the escape, and thus, it is difficult to fully evaluate an individual’s emergency escape capability. The formula of emergency escape capability in this paper is only applicable to this study’s virtual subway fire escape system and can provide a reference for similar research. Whether it is applicable to other system models remains to be further verified. Moreover, regarding the experimental results on influencing factors on an individual’s emergency escape capability, only one single prototype of Zijing Mountain subway station was used in this paper, which may reduce the reliability on the research results. Other prototype studies will be carried out to draw more credible conclusions in the future.(3)ParticipantsParticipants were all undergraduates (ranging from 19 to 22 years old) from Zhengzhou University in China; the sample size is a little small. Considering the prevalence of COVID-19 and even Delta variant everywhere, due to the limitations of the experimental conditions, we only chose undergraduates in our university as participants. However, choosing participants to be consistent with the actual composition of subway passengers would draw more credible conclusions. In our future studies, we will expand subject sample groups and size, and choose participants to be consistent with the real composition of subway occupants as much as possible. We will also explore other factors that affect an individual’s emergency escape capability, like different age groups, people with wheelchairs, hearing impaired, medication, substance use, tiredness, drunkenness, or people traveling in groups, or other individual characteristics, etc., in our future research.(1)A calculation formula of emergency escape capability is proposed, which can evaluate an individual’s emergency escape capability quantitatively in subway virtual fire escape.(2)p < 0.05).Gender is a factor affecting an individual’s emergency escape; men’s emergency escape capability is better than women’s ((3)p > 0.05). Although there is no significance in emergency escape capability in DISC, the mean emergency escape capability in compliance personality type is the best, and the mean emergency escape capability in influence personality type is the worst. This provides thoughts for targeted training on different personality types of occupants in subways in the future.There is no significance in emergency escape capability in DISC personality type ((4)p < 0.01), which indicated that participants were under stress during their virtual fire escape. Moreover, there is a significant negative correlation between an individual’s emergency escape capability and LF_increase_rate (p < 0.05), and a very significant negative correlation between an individual’s emergency escape capability and LF/HF_increase_rate (p < 0.01). This indicates that the greater the increase rate of LF and LF/HF, the smaller the emergency escape capability, while an increase of LF and LF/HF represents the person being in a stressful state, which may reflect that excessive stress is not conducive to emergency escape. Therefore, during the whole emergency evacuation process, subway management departments should take various emergency management and emergency evacuation measures to maintain occupants’ emotional stability and avoid their excessive stress.During virtual fire escape vs. baseline, Mean_SC and Mean_HR both increased very significantly p < 0.01), which indicates that the more familiar with the subway station, the higher the emergency escape capability is. This provides a way to improve an individual’s emergency escape capability by becoming familiar with the station consciously, including familiarity with various escape routes and emergency exits after entering the subway station, etc.There is a very significant negative correlation between an individual’s emergency escape capability and the degree of familiarity with the Zijing Mountain subway station  in this experiment, the following conclusions were drawn:


Rationalizing the identification of hidden similarities across the repertoire of druggable protein cavities remains a major hurdle to a true proteome-wide structure-based discovery of novel drug candidates. We recently described a new computational approach (ProCare), inspired by numerical image processing, to identify local similarities in fragment-based subpockets. During the validation of the method, we unexpectedly identified a possible similarity in the binding pockets of two unrelated targets, human tumor necrosis factor alpha (TNF-α) and HIV-1 reverse transcriptase (HIV-1 RT). Microscale thermophoresis experiments confirmed the ProCare prediction as two of the three tested and FDA-approved HIV-1 RT inhibitors indeed bind to soluble human TNF-α trimer. Interestingly, the herein disclosed similarity could be revealed neither by state-of-the-art binding sites comparison methods nor by ligand-based pairwise similarity searches, suggesting that the point cloud registration approach implemented in ProCare, is uniquely suited to identify local and unobvious similarities among totally unrelated targets.The online version contains supplementary material available at 10.1186/s13321-021-00567-3. Among the many possible approaches to structure-based drug design , 2, infe, scaling, rotation and translation) that aligns two point clouds ,Contrarily to our prediction, no thermophoresis signal could be detected in presence of nevirapine Fig. C indicatIn absence of X-ray structures of TNF-α bound to efavirenz and delavirdine, we cannot rule out the possibility that both inhibitors bind to a different pocket than that highlighted in the current computational study. This hypothesis is however very unlikely for two reasons: (i) no other cavity than that occurring at the inner core of the multimeric TNF-α could be detected among the currently existing 33 structures available in the Protein Data Bank; (ii) all non-covalent small molecular weight inhibitors co-crystallized with TNF-α dimeric or trimeric forms –35 are eWe should recall here that none of the HIV-1 RT inhibitors has been optimized for binding to TNF-α and is directly repurposable for treating TNF-α -dependent autoimmune disorders. However, we do think that efavirenz may be optimized to a much more potent HIV-1 RT inhibitor by following a strategy similar to that reported to modify the 22 µM TNF-α inhibitor UCB-6876 to a 9 nM lead  when compared to the ProCare algorithm . Extending 2D fingerprint comparisons to additional 2,361 HIV-1 RT inhibitors  but predicted to share local similarities according to our recently-developed ProCare point cloud registration method. The computational prediction was verified by microscale thermophoresis experiments evidencing the micromolar binding of some but not all HIV-1 RT non-nucleoside inhibitors to human soluble TNF-α. Interestingly, the ProCare prediction could not be revealed by other state-of-the-art cavity or ligand similarity search methods. Point cloud registration, a computational method frequently used for digital image processing in robotics and medical imaging, enables the detection of subtle and local protein similarities thanks to a powerful description of subpocket microenvironments. The ProCare method appears as a promising idea generator for drug repurposing and fragment-based ligand design since it is able to pick starting ligands at a proteomic scale.https://www.rcsb.org) [The recently described asymmetric structures of the human TNF-α trimer bound to different inhibitors were retrieved from the RCSB Protein Data Bank (PDB) homepage (csb.org)  using thcsb.org) . The PDBcsb.org)  v.4.0, tcsb.org)  (cavity_https://www.rcsb.org) [http://bioinfo-pharma.u-strasbg.fr/scPDB) [Starting from the PDB structure 1VRT as a reference, a search was performed in the RCSB PDB (csb.org)  to retrir/scPDB)  and for r/scPDB)  v4.0. Thr/scPDB)  fragmentr/scPDB)  and the r/scPDB)  cavity phttps://www.ebi.ac.uk/chembl) by querying the general keyword ‘reverse transcriptase’ and retaining ChEMBL target identifiers  corresponding to HIV-1 RT. Ligands with a measured sub-micromolar half-maximal inhibitory concentration (IC50) against the HIV1-RT single target were defined here as inhibitors  v.2019.03.4.0 to remove redundancy.Bioassay information were first retrieved from the ChEMBL  dataset http://bioinfo-pharma.u-strasbg.fr/scPDB) [Ligands coordinates from the sc-PDB (r/scPDB)  v.2016 ar/scPDB) . FragmenProCare  v.0.1.1 FuzCav , an aligG-LoSA  v.2.2 ishttp://ligand-expo.rcsb.org/) and prepared according to the KRIPO procedure (https://github.com/3D-e-Chem/kripo). Then KRIPO [PDB ligands structural information were downloaded from Ligand Expo  was computed for each prepared PDB structures with the default parameters referenced in the manual (3.0 Å to the ligand). ProBiS  requiresFor each entry, the list of natural amino acids in the ‘SITE’ mol2 files were provided as input. SiteAlign  v.4.0 deShaper  v.1.0 ushttp://www.rdkit.org) python package v.2019.03.4.0 using default parameters (radius = 2 for the Morgan fingerprints). The Tanimoto coefficients of the pairwise TNF-α ligands/HIV-1 RT ligands fingerprints comparison were reported. Cut-off values of 0.30 (Morgan fingerprints) and 0.65 (MACCS keys) were used to discriminate chemically similar from dissimilar ligands.Morgan fingerprints on the one hand, and 166 public MACSS keys on the other hand were computed on the PDB ligands (sdf format) and ChEMBL ligands (SMILES strings) with RDKit . A TanimotoCombo cut-off value of 1.5 was used to discriminate chemically similar from dissimilar ligands.TNF-α X-ray structure 6OOZ was prepared as described above (see TNF-α structures). 6OOZ co-crystallized ligand on the one hand, delavirdine, efavirenz and nevirapine as well as their main fragments on the other hand were drawn with MarvinSketch v.16.10.17  and saved into 2D sdf format. They were ionized with Filter v.2.5.1.4  using customized parameters . Soluble human TNF-α  was purchased from GenScript (http://www.genscript.com).Nevirapine , efavirenz  and delavirdine mesylate  were purchased from Selleck Chemicals . Thermophoresis was measured at 25 °C with 40% LED power and 20%, 40% and 80% MST power using a Monolith NT.115 (NanoTemper Technologies GmbH). Data were analyzed in the NT Analysis software version 1.5.41 (NanoTemper Technologies GmbH).Human TNF-α was labeled using the RED-NHS 2nd generation labeling kit (NanoTemper Technologies GmbH) using a protein concentration of 10 µM and a molar dye-to-protein ratio ~ 3:1. A label/protein ratio of 0.4 was determined using photometry at 650 and 280 nm. Compounds efavirenz, delavirdine and nevirapine were initially dissolved in DMSO to afford stock solutions of 10 mM. These were then diluted to initial concentrations of 260 μM into 20 mM K-phosphate pH 7.4, 150 mM NaCl ensuring a final concentration of DMSO of 2.6%. These compounds were serially diluted 2:1 in buffer 20 mM K-phosphate pH 7.4, 150 mM NaCl, 2.6% DMSO producing ligand concentrations ranging from 260 µM to 594 nM (16 titration points). For MST measurements, each ligand dilution was mixed with 1 volume of fluorescently-labelled TNF-α at 680 nM in 20 mM K-phosphate pH 7.4, 150 mM NaCl, 0.02% Tween-20, which leads to a final concentration of TNF-α of 340 nM and final ligand concentrations at half of the ranges above. The final buffer is 20 mM K-phosphate pH 7.4, 150 mM NaCl, 0.01% Tween-20 and 1.3% DMSO. After a 15-min incubation at room temperature in the dark, followed by centrifugation at 13,000Additional file 1: Fig. S1: Receiver operating characteristic (ROC) curves derived from ProCare similarity scores. Fig. S2: ProCare alignment of efavirenz main fragment subpocket onto TNF-α trimer pocket. Fig. S3: Contributions of the eight pharmacophoric features to the ProCare similarity score between HIV-1 RT and TNF-α. Fig. S4: Non-covalent interactions between efavirenz and HIV-1 RT, and between UCB-5307 and TNF-α trimer. Fig. S5: Manual fragmentation of delavirdine in three fragments (#1 to #3). Table S1: sc-PDB subpockets sorted by decreased ProCare similarity to the inner cavity of human TNF-α. Table S2: PDB entries describing non-nucleoside inhibitors bound to HIV-1 reverse transcriptase. Table S3: Comparison of delavirdine subpockets, resulting from manual fragmentation, with TNF-α trimer pockets. Table S4: Dissociation constant (KD) of three HIV-1 RT inhibitor binding to human soluble TNF-α, according to MST experimental conditions. Table S5: CHEMBL entries describing HIV-1 RT non-nucleoside inhibitors. Table S6: Customized rules for OpenEye Filter ionization.


TNF, IL genes, TNFAIP3), transporters , receptors  and associated proteins , as well as other genes implicated in the pathogenesis of psoriasis  can be used in the future as predictive markers of treatment response and/or toxicity with biological therapies in patients diagnosed with moderate-to-severe psoriasis, tailoring treatment to the individual patient.Psoriasis is a chronic inflammatory skin pathology of autoimmune origin and unknown etiology. There are various therapies for treating it, including a wide range of biopharmaceuticals indicated in moderate-to-severe psoriasis. Depending on their therapeutic target, they are classified as tumor necrosis factor inhibitors (anti-TNF) or cytokine inhibitors . Although they have proved effective and safe, in clinical practice, many patients show a short- and long-term suboptimal response and even varying degrees of toxicity. This variability in response may be influenced by genetic factors, such as polymorphisms in the genes involved in the pathological environment, metabolism or mechanism of action of the drug that could affect the effectiveness and toxicity of biological therapies. This review assesses pharmacogenetic studies of the impact of genetic factors on response to biopharmaceuticals and toxicity in patients diagnosed with moderate-to-severe psoriasis. The results suggest that polymorphisms detected in the HLA genes, in genes that encode cytokines ( Psoriasis is a chronic and recurrent inflammatory autoimmune skin disease with a worldwide prevalence of up to 8.5% in adults and 2.1% in children . FurtherHLA-Cw*06 alleles  have been described as the first-factor risk of psoriasis. Remarkably, HLA-Cw*06 was found to mediate autoimmunity against melanocytes through the ability of its protein product to present ADAMTS-like protein 5 (ADAMTSL5). In fact, this complex is recognized by epidermal CD8+ T cells, which directly target melanocytes and produce inflammatory cytokines (such as TNF-α and IL-17) that, in turn, are able to alter melanocyte functions and proliferation, leading to dysregulation of skin homeostasis . It . It 8]. eostasis .KRT1, RT6A, KRT6B, KRT10, KRT16 and KRT17). Moreover, altered collagen structure and function due to relevant mutations can affect all tissues or organs and cause various pathological phenotypes. Specifically, two genetic variants of the collagen (COL) genes, COL8A1 and COL8A2, have been shown to be associated with the extracellular matrix remodeling and angiogenesis that occurs in psoriasis. In addition, the impact of genetic variants in COL may be associated with the development of psoriatic arthritis in patients with cutaneous psoriasis  . Sp. Sp90]. = 0.022) . HLA-A rs9260313/TRAF3IP2 rs13190932 and HLA-Cw*06/LCE3C_LCE3B del/ins haplotypes, together with the HLA-B/MICA rs13437088 and HLA-C rs12191877, rs1048554 and rs610604 polymorphisms, have shown an association with the response to anti-TNF drugs. However, the presence of the HLA-Cw*06 alleles has been shown to be associated with response to UTK but not to anti-TNF drugs. In addition, associations with response to anti-TNF drugs in patients diagnosed with moderate-to-severe psoriasis have been found for polymorphisms of the following genes: TNF , IL6 (rs1800795), IL12B (rs2546890), TNFAIP3 (rs6920220), PDE3A-SLCO1C1 (rs11045392-rs3794271), TNFRSF1A (rs1061622), CD84 (rs6427528), FCGR2A (rs1801274), FCGR3A (rs396991), IL17RA (rs4819554), IL23R (rs11209026), TLR2 (rs4696480 and rs11938228), TLR9 (rs352139), PGLYRP4–24 (rs2916205), CDKAL1 (rs6908425), CARD14 (rs11652075), PTTG1 (rs2431697), MAP3K1 (rs96844), ZNF816A (rs9304742), GBP6 (rs928655), CTNNA2 (rs11126740) and HTR2A (rs6311). Furthermore, polymorphisms of the following genes have shown an association with response to UTK: IL12B (rs3213094), TNFAIP3 (rs610604), LY96 (rs11465996), TIRAP (rs8177374) and TLR5 (rs5744174), and the IL1B rs1143623 and rs1143627 and IL17F rs763780 polymorphisms have proved to be associated with susceptibility to developing toxicity and paradoxical psoriasis due to anti-TNF drugs.There is a wide range of biologics indicated for moderate-to-severe psoriasis that have proved effective and safe; however, certain patients do not obtain the expected effect in the short or long term and experience various degrees of toxicity. This variability in short- and long-term response and in toxicity may be due to genetic factors. Precision medicine research has assessed the influence of polymorphisms of genes involved in the pathological environment of the disease, metabolism or mechanism of action on the efficacy of these drugs. Specifically, the allelic variants of HLA genes have been extensively studied, but the results are contradictory. The In general, we have summarized the genetic polymorphisms that have been shown to play a significant role in the effectiveness of biopharmaceuticals in moderate-to-severe psoriasis. However, this review has several limitations. First, many of the existing studies have small sample sizes, which makes it difficult to reach a definitive interpretation of the role of these genes in response to treatment with biopharmaceuticals. Second, the studies show a lack of uniformity in their definitions of the treatment response and follow-up period, which impacts the possibility of generalizing from the results. Third, most of the studies have analyzed the response to anti-TNF drugs as a group, despite the fact that they have different molecular structures and degrees of efficacy, giving rise to the opacity of results. Finally, new targets have been introduced in the treatment algorithm for moderate-to-severe psoriasis, which has proved more effective than drugs than inhibit TNF or IL-12, IL-17 and IL-23. However, pharmacogenetic markers for these drugs are not yet available. Consequently, new studies are needed to look for specific biomarkers for each drug with a uniform definition of response, longer-term follow-up and larger sample sizes to confirm these results.TNF, IL genes, TNFAIP3), transporters , receptors  and associated proteins , as well as other genes implicated in the pathogenesis of psoriasis  can be used in the future as predictive markers of treatment response and/or toxicity with biological therapies in patients diagnosed with moderate-to-severe psoriasis, tailoring treatment to the individual patient.In conclusion, the results suggest that polymorphisms detected in the HLA genes, in genes that encode cytokines (


As magnetic resonance imaging (MRI) signs of normal pressure hydrocephalus (NPH) may precede clinical symptoms we sought to evaluate an algorithm that automatically detects this pattern.A support vector machine (SVM) was trained in 30 NPH patients treated with ventriculoperitoneal shunts and 30 healthy controls. For comparison, four neuroradiologists visually assessed sagittal MPRAGE images and graded them as no NPH pattern, possible NPH pattern, or definite NPH pattern.Human accuracy to visually detect a NPH was between 0.85 and 0.97. Interobserver agreement was substantial (κ = 0.656). Accuracy of the SVM algorithm was 0.93 and AUROC 0.99. Among 272 prespecified regions, gray matter and CSF volumes of both caudate, the right parietal operculum, the left basal forebrain, and the 4th ventricle showed the highest discriminative power to separate a NPH and a no NPH pattern.A NPH pattern can be reliably detected using a support vector machine (SVM). Its role in the work-up of asymptomatic patients or neurodegenerative disease has to be evaluated. Normal pressure hydrocephalus (NPH) is a brain disorder in which excess cerebrospinal fluid (CSF) accumulates in the subarachnoid and ventricular spaces. It is defined as increased CSF volume without continuous elevation of the intracranial pressure, whereas the complete pathomechanism is still unclear . A resulIn some cases, NPH is caused by other brain disorders such as a tumor, head injury, hemorrhage, infection or inflammation. Yet in most cases, the cause of excess fluid remains unknown. Studies indicate that the development of NPH underlies a prolonged process since radiographic findings show volumetric changes 3 or more years before the onset of clinical symptoms [Despite ongoing efforts, neither the pathophysiology of NPH nor the mechanics of the improvement after standard treatment with surgical CSF shunting are completely understood. The diagnosis of NPH is dependent on several clinical and radiological factors and is complicated by a variable degree of symptom presentation as well as neurodegenerative comorbidities . Given tImaging used for the diagnosis of neurodegenerative diseases serves two functions: it should reveal treatable causes and contribute to the etiological differentiation of primary dementia disorders . TreatabIn imaging, the most reliable sign for detecting NPH is a disproportional enlargement of the ventricles and the Sylvian fissure  in comparison to the constricted CSF spaces in the high convexities of the brain . While vThe aim of this study was to implement a machine learning tool that automatically identifies NPH-typical MRI features (NPH pattern) on routinely acquired MRI sequences.Patients: presurgical MRI scans of 30 NPH patients, who had been treated with a ventriculoperitoneal shunt were retrospectively compared to an age and gender-matched group of 30 healthy controls (HC). Surgery had been performed between 2010 and 2017, and patients re-evaluated several times after shunt implantation thereafter. We screened our neurosurgical database retrospectively for patients first time receiving a CSF shunt due to a NPH between 2010 and 2017. As a second step, we included those having undergone 3D T1-weighted MRI scans.3). They were asked to assess imaging features, such as the DESH sign, THC and small callosal angle and to finally grade them on a 3-point scale . Anonymized imaging data of patients and controls were shown in a randomized order. In addition, MRI reports were retrospectively reviewed whether NPH features were described or the condition was dismissed, e.g. as unspecific ventriculomegaly.Readings: two senior (26 and 19 years of experience) and two junior neuroradiologists (4 and 2 years of experience) independently assessed the presence of a NPH pattern on a 3D sagittal magnetization prepared rapid acquisition gradient recalled echo (MP-RAGE) sequence with isotropic voxels (1 × 1 × 1 mmhttp://www.neuromorphometrics.com/) were extracted as a direct input into the SVM . Fivefold cross-validation was performed. The SVM results are given as a prediction score from 0 to 1. An example for an analysis is displayed in Fig. Machine learning: for NPH identification, a linear support vector machine (SVM) was employed . A SVM iStatistical analysis: SVM performance was compared to human ratings in terms of correctly predicting NPH cases. Standard metrics for binary classification such as accuracy, sensitivity, specificity and negative prediction value were calculated. A receiver operating characteristic (ROC) analysis displayed sensitivity and specificity of SVM in identifying NPH patients. Interobserver agreement was assessed by Fleiss’ κ .A total of 30 patients were included in the NPH group and 30 HC served as control group. Clinical characteristics are displayed in Table Patients were clinically re-evaluated directly after and 3 months after surgery (mean 57 days). One patient was lost in follow-up and another patient died after surgery  due to acute respiratory distress syndrome. After shunting, 20 patients  showed a marked improvement of NPH symptoms. In two patients, improvement was unclear, six patients did not benefit.p < 0.001).Human readings are displayed in Table SVM performance: with a prediction score of 0.42, acceptable performance was achieved with one false negative and three false positive results. Human readings and SVM results are compared in Table With respect to the discriminative power of the 272 regions defined in the probabilistic brain atlas, the following 5 regions served best to separate NPH and healthy controls: right caudate, right parietal operculum, left basal forebrain, left caudate and 4th ventricle  in old patients and rising numbers of MRI scans in this group, one can presume an increased incidence of a NPH pattern. Since the NPH pattern may precede clinical symptoms, automatic detection during a presymptomatic stage could be a promising tool for an early diagnosis. Trained machines such as the presented support vector machine (SVM) might simplify diagnosis with no apparent loss of reliability compared to human performance.


Objective: Accumulating evidence suggested that resveratrol (RES) could protect against adverse cardiac remodeling induced by several cardiovascular diseases. However, the role of RES in the setting of heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain understood. This study was to determine whether RES could ameliorate HFpEF-induced cardiac remodeling and its mechanisms.Methods:In vivo, C57BL/6 mice served as either the sham or the HFpEF model. The HFpEF mice model was induced by uninephrectomy surgery and d-aldosterone infusion. RES  or saline was administered to the mice for four weeks. In vitro, transforming growth factor β1 (TGF-β1) was used to stimulate neonatal rat cardiac fibroblasts (CFs) and Ex-527 was used to inhibit sirtuin 1 (Sirt1) in CFs. Echocardiography, hemodynamics, western blotting, quantitative real-time PCR, histological analysis, immunofluorescence, and ELISA kits were used to evaluate cardiac remodeling induced by HFpEF. Sirt1 and Smad3 expressions were measured to explore the underlying mechanisms of RES.Results: HFpEF mice developed left ventricular hypertrophy, preserved ejection fraction, diastolic dysfunction, and pulmonary congestion. Moreover, HFpEF mice showed increased infiltration of neutrophils and macrophages into the heart, including increased interleukin (IL)-1β, IL-6, and TNF-α. We also observed elevated M1 macrophages and decreased M2 macrophages, which were exhibited by increased mRNA expression of M1 markers  and decreased mRNA expression of M2 markers  in HFpEF hearts. Moreover, HFpEF hearts showed increased levels of intracellular reactive oxygen species (ROS). Importantly, HFpEF mice depicted increased collagen-I and -III and TGF-β mRNA expressions and decreased protein expression of phosphorylated endothelial nitric-oxide synthase (p-eNOS). Results of western blot revealed that the activated TGF-β/Smad3 signaling pathway mediated HFpEF-induced cardiac remodeling. As expected, this HFpEF-induced cardiac remodeling was reversed when treated with RES. RES significantly decreased Smad3 acetylation and inhibited Smad3 transcriptional activity induced by HFpEF via activating Sirt1. Inhibited Sirt1 with Ex-527 increased Smad3 acetylation, enhanced Smad3 transcriptional activity, and offset the protective effect of RES on TGF-β–induced cardiac fibroblast–myofibroblast transformation in CFs.Conclusion: Our results suggested that RES exerts a protective action against HFpEF-induced adverse cardiac remodeling by decreasing Smad3 acetylation and transcriptional activity via activating Sirt1. RES is expected to be a novel therapy option for HFpEF patients. Heart failure with preserved ejection fraction (HFpEF) has been gradually increasing due to increasing aging population; it was reported that HFpEF now accounts for nearly half (39–72%) of all heart failure patients . AdverseResveratrol  (RES)  is a natSirtuin 1 (Sirt1) deacetylase, a class III histone deacetylase, could deacetylate various proteins . RecentlAccordingly, we theorized that RES can effectively protect against HFpEF-induced cardiac inflammation, oxidation, and fibrosis by activating Sirt1.RES was purchased from Sigma , and d-aldosterone was purchased from Sigma . Ex-527 was purchased from Sigma (#E7034).via osmotic mini-pumps   were supplied by the animal experiment center of Tongji Medical College, Huazhong University of Science and Technology. Mice were allowed 1 week to acclimatize to a stable environment before experiments began. Mice were individually housed in plastic cages with bedding, ad libitum food, and tap water. The cages were maintained at a temperature of 22 ± 2°C and a 12:12 h light/dark cycle. Mice were randomly assigned to four groups: sham with vehicle (sham-vehicle), sham with RES (sham-RES), HFpEF with vehicle (HFpEF-vehicle), and HFpEF with RES (HFpEFRES). All mice underwent uninephrectomy and received a continuous infusion of either saline (sham) or d-aldosterone (0.15mg/h) (HFpEF) for 4 weeks  States) . Twenty- States)   . All expIsoflurane (1.5%) was used to anesthetize; then, echocardiography was conducted using a MyLab 30CV ultrasound machine with a 10 MHz linear array ultrasound transducer. M-mode images of the left ventricle at the papillary muscle level were recorded, and then left ventricular ejection fraction (EF) and fractional shortening (FS) were measured . HemodynHearts were isolated and arrested in 10% KCl solution. Then, after fixation with 4% paraformaldehyde for 5 days, the hearts were embedded in paraffin and sliced into approximately 5 µm sections. Subsequently, the sections were stained with wheat germ agglutinin (WGA) for the determination of the cardiomyocyte size; 10 different areas per heart were evaluated.For immunofluorescence detection, the sections with a thickness of 10 μm were incubated with the primary antibody against Ly6G , F4/80 , CD80 , and CD206 ; afterward, sections were incubated with FITC-conjugated anti-rabbit whole IgG and Texas Red–conjugated anti-mouse whole IgG. The stained sections were observed and photographed under a microscope (×200) of 20 random fields of view of each heart sample.The prepared serum was assayed for IL-1β (#EMC001b.96), IL-6 (#YIF-LF-EK0270), and TNF-α (#EMC102a.96) using the Quantikine Sandwich ELISA Kit following the manufacturer’s instructions .Briefly, hearts were transversely cut close to the papillary muscles, fixed in 10% phosphate-buffered formalin, and embedded in paraffin. The paraffin-embedded hearts were sliced into 5 μm sections that were stained with picrosirius red (PSR) to quantify cardiac fibrosis. The stained sections were observed and photographed under a microscope (×200) of 15 random fields of view of each heart sample. The left anterior free-wall fields were analyzed and averaged to calculate the collagen fibrosis in each heart.To detect ROS, DHE staining was conducted. Briefly, cryosections of fresh heart samples were stained with DHE  for 30 min at 37°C to detect ROS production. Pictures were taken with an OLYMPUS DX51 fluorescence microscope . Furthermore, fresh heart samples were homogenized on ice and centrifuged at 3,000 rpm for 15 min. After that, the supernatants were collected to measure the activities of superoxide dismutase  and catalase  and the content of glutathione  by commercial kits according to the manufacturer’s instructions. The stained sections were observed and photographed under a microscope (×200) of 20 random fields of view of each heart sample.−1) and trypsin (0.1%). Then, cells were obtained and plated for 1.5 h at 37°C until the CFs adhered to the wall of the plate. After discarding the pre-seeding medium containing cardiomyocytes and unattached cells, the relatively pure CFs were collected. CFs were then cultured in DMEM/F12  with 10% fetal bovine serum  at 37°C in a humidified incubator with 5% CO2. All CFs were treated within three passage cultures, but only CFs prior to the third passage could be used in our study. After serum starvation for 24 h and synchronization, CFs were randomly treated with TGF-β  and RES (25 μm) for 24 h. The Sirt1 expression in CFs was blocked by Ex-527 (10 µM).Neonatal rat cardiac fibroblasts (CFs) were isolated from the newborn Sprague Dawley rats (1–3 days) as previously reported . BrieflyTotal RNA was prepared using TRIzol reagent (Invitrogen) from heart samples or cell lysates. Two micrograms of RNA were reverse transcribed into cDNA with random primers using a Transcriptor First Strand cDNA Synthesis Kit. Real-time PCR was performed using LightCycler 480 SYBR Green 1 Master Mix. The PCR conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 15 s and a final step at 72°C for 15 s. The level of each gene was measured in triplicate in at least three independent experiments. Calculations of relative mRNA expression levels were conducted using the ΔΔCT method. GAPDH was used as an internal reference. The mRNA data were normalized to GAPDH and the sham-vehicle and PBS-treated groups. All primer details are listed in Total protein was extracted and separated on SDS-PAGE . Then, proteins were transferred onto PVDF membranes . The membranes were incubated overnight at 4°C with the primary antibody of IL-1β , IL-6 , TNF-α , TGF-β1 , p-eNOS , eNOS , Sirt1 , and GAPDH . The membranes were then incubated for 2 h at room temperature with HRP-conjugated secondary antibodies . The western blot film images were scanned and analyzed using ImageJ . The total protein levels were normalized to GAPDH and the sham-vehicle group.Immunoprecipitation analysis was performed as described previously . Heart tEqual amounts of total protein were used for each experimental condition. The reagents from a Sirt1 Assay Kit  were used according to the manufacturer’s instructions. Sirt1 deacetylase activity was measured using fluorescence intensity signals at 450 nm  using a microplate fluorimeter. Experimental values are represented as activity relative to that observed in the sham-vehicle group or PBS group.The transcriptional activity of Smad3 was measured using a luciferase reporter gene assay. Briefly, the CFs were transfected with a firefly luciferase reporter plasmid Smad3-CAGA-Luc mixed with an internal control pRL-TK vector (Promega) as described previously . The trat-test or chi-square test. The differences between multiple groups were analyzed with two-way analysis of variance (ANOVA) with Tukey’s test. p < 0.05 was considered statistically significant.All data in the tables and figures are expressed as mean ± SD and were analyzed using GraphPad Prism 8.0 software. Differences between two groups were assessed using Student’s p < 0.05, p < 0.05, p < 0.05). RES treatment significantly decreased the HW/BW ratio in the HFpEF-RES group than the HFpEF-vehicle group (p < 0.05) and obviously reversed HFpEF-induced LVH, which was further verified by the mRNA levels of LVH markers  is an important characteristic of HFpEF. We initially checked the LVH in HFpEF mice which was measured by cardiomyocytes’ size; WGA staining showed that the cross-sectional area of LV cardiomyocytes in the HFpEF-vehicle group was significantly larger than that in the sham-vehicle group (p < 0.05), and RES significantly decreased the release of HFpEF-induced pro-inflammatory cytokines  , and RES markedly decreased IL-1β, IL-6, and TNF-α mRNA expressions. Similar to the results of RT-PCR analysis, western blot analysis depicted increased IL-1β, IL-6, and TNF-α protein expressions in HFpEF hearts, and RES significantly decreased IL-1β, IL-6, and TNF-α protein expressions . In addition, further detection revealed increased activities of CAT, SOD, and GSH in HFpEF hearts compared with sham hearts  protein expression is associated with increased cardiac fibrosis , and RESin vitro. Ex-527 was used to decrease the Sirt1 expression. As shown in collagen-I and -III and TGF-β was significantly increased when treating CFs with TGF-β; co-treating CFs with TGF-β and RES significantly decreased the mRNA expression of cardiac fibrosis markers collagen-I and -III and TGF-β. And inhibited Sirt1 with Ex-527 co-treated with TGF-β and RES offset the protective role of RES in TGF-β–induced cardiac fibrosis.The previous study reported that RES is a Sirt1 activator . We assevia activating Sirt1; and 6) inhibited Sirt1 with Ex-527 offset the protective role of RES in TGF-β–induced cardiac fibroblast–myofibroblast transformation. In summary, these findings indicate that RES might be a potent drug that exerts protective effect on HFpEF-induced cardiac remodeling by inhibiting inflammatory responses, modulating macrophage polarization, reversing oxidation, activating Sirt1, and suppressing Smad3 acetylation, thereby reducing cardiac fibrosis and cardiac stiffness  RES could significantly ameliorate HFpEF-induced LVH, diastolic dysfunction, and pulmonary congestion without impairing the LVEF; 2) RES treatment decreased the infiltration of inflammatory cells and regulated M1/M2 macrophage polarization in the heart; 3) RES reversed HFpEF-induced superoxide production, restored HFpEF-induced extracellular matrix deposition, and increased the phosphorylated eNOS expression; 4) in addition, RES also inhibited the TGF-β/Smad3 pro-fibrotic signaling pathway; 5) RES decreased Smad3 acetylation and inhibited Smad3 transcriptional activity tiffness . These oAccumulating evidence suggested that RES exerts anti-inflammatory effect on several settings of cardiovascular diseases except HFpEF. This time, we aimed to test the cardioprotective effect of RES on cardiac inflammation and the potential mechanisms. Previous experiments revealed that myocardial inflammatory response plays a pivotal role in the development of HFpEF. In an animal experiment, circulating levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β were markedly increased in HFpEF mice compared to sham mice . FurtherInappropriate macrophage polarization and excessive inflammatory factors are reported to be linked with increased macrophage infiltrates in the impaired myocardium . HoweverExcessive myocardial ROS release plays a vital role in HFpEF. It is likely a systemic inflammatory state that can both produce free radicals and blunt cellular anti-oxidant capacity. Moreover, excessive ROS could lead to collagen uncoupling and the formation of superoxide anions, reduce NO bioavailability, and ultimately impair cardiac function . It was The NO-cGMP-PKG pathways were reported to be a potentially promising therapeutic target for HFpEF . CardiacThe inflammation-driven stimulation of TGF-β/Smad3 signaling is essential for the transdifferentiation of fibroblasts into myofibroblasts and collagen deposition . FurtherSirtuin 1 (Sirt1) deacetylase is a class deacetylase, which could deacetylate various proteins . EmerginThus, this study provides evidence that HFpEF results in a systemic pro-inflammatory state, causes excessive ROS production, leads to downregulation of eNOS and myocardial stiffness, inhibits Sirt1 activation, increases Smad3 acetylation, activates the TGF-β signaling pathway, and eventually enhances myocardial fibrosis . By inhiIn conclusion, it is clear that the mechanisms responsible for the beneficial effects of RES on HFpEF-induced cardiac remodeling are completely complex and various. The present study provides novel insights into the fact that the nutritional agent RES may exert protective effect in the setting of HFpEF by combining anti-inflammatory, anti-oxidant, and anti-fibrotic actions coupled with improved cardiac stiffness.


ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell.Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression.In this study the course of three different Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain.Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.The online version contains supplementary material available at 10.1186/s12936-021-03832-w. Plasmodium falciparum parasites, the causative agent of malaria, are developing resistance to a number of front-line anti-malarial drugs (such as artemisinin), which has the potential to severely impact malaria control in the future [Malaria is a major public health problem in developing countries, primarily in sub-Saharan Africa and Asia . There we future , 4. ConsATP2B4) to have a very strong association with resistance and severity of malaria infection [ATP2B4 as a significant resistance loci for severe malaria [ATP2B4 across different populations and studies suggests a major role for the gene product of ATP2B4 in modulating the severity of Plasmodium infection.Understanding the critical host pathways that control the susceptibility and resistance of individuals to malaria in endemic countries may afford novel strategies to treat severe malaria. A recent human genome wide association study (GWAS) in Kenya identified single nucleotide polymorphisms (SNPs) in the human plasma membrane calcium ATPase 4 (PMCA4) gene  RBCs has been associated with inhibition of PMCA channel activity and increased intracellular Ca2+ concentrations [2+ concentrations can lead to RBC dehydration [P. falciparum parasites are unable to invade and grow as efficiently in dehydrated RBCs [PMCAs are ATP-driven and calmodulin-dependent calcium pumps that eject calcium from the cell cytoplasm to the extra-cellular compartment . There anM range . Thus, inM range , 11. Nottrations . The incydration , and theted RBCs . TargetiPmca4 gene (PMCA4−/− mice) were infected with different species of murine Plasmodium parasites. Surprisingly, whilst abrogation of PMCA4 influences the morphology and biology of RBCs, it did not substantially affect peripheral parasite levels during Plasmodium yoelii, Plasmodium chabaudi or Plasmodium berghei infections. Inhibition of PMCA4 did, however, slightly increase the resistance of mice to experimental cerebral malaria (ECM). The results suggest that PMCA4 may have an effect on the development of severe malaria disease but raise important questions on the mechanisms involved.In this study to directly assess the role of PMCA4 in modulating the course of blood stage malaria, mice with a systemic genetic knockout of the All animal work was approved following local ethical review by the University of Manchester (UoM) Animal Procedures and Ethics Committees and was performed in strict accordance with the U.K Home Office Animals (Scientific Procedures) Act 1986 (approved H.O Project Licence P8829D3B4).−/−) mice and wild type (WT) littermates were used in this study. Generation of PMCA4−/− mice was described in our previous publication [−/− mice are generally normal, as defined by whole body phenotype analysis [Plasmodium chabaudi used was the non-lethal AS strain obtained from Dr K.N. Brown , London) [Plasmodium yoelii NL strain was obtained from Dr Brian De Souza  [Plasmodium berghei ANKA used was the clone cl15cy1 as described in [P. chabaudi AS, P. yoelii NL or P. berghei ANKA parasites were passaged once in C57BL/6 mice before experimental mice were infected via intravenous (i.v.) injection of 104 parasitized red blood cells (pRBC) of one of the Plasmodium species. The course of infection was monitored by assessing peripheral parasitaemia via microscopic examination of Giemsa-stained thin blood smears, by calculating mouse weight on specified days of infection (compared to starting weight at initiation of experiment), and by quantifying erythrocyte numbers in peripheral blood (RBC /ml) by microscopy using a C-Chip disposable haemocytometer (Cambridge Bioscience). ECM was graded during P. berghei ANKA infection as previously described [Plasmodium berghei infected mice were euthanized when they reached stage 4/5. To examine the permeability of the blood brain barrier (an important event during ECM), mice were injected i.v. with 200 ml of 1% Evans blue  on day 6 of P. berghei infection. After one hour, mice were culled and intra-cardiac whole-body perfusion with 15 ml of PBS was performed, following which brains were removed.PMCA4 global knock out  . The Pla London) , whereasribed in . Dependiescribed : 1 = no For peripheral blood analysis, mice were anesthetized using 2.5% isoflurane and blood was collected from the jugular vein. Blood samples were analysed within 6 h after collection using an automated blood analyser (Sysmex XT-2000iV) at room temperature. Blood analysis was carried out in 2 batches using a mouse profile.−/− and WT mice under anaesthesia with 2.5% isoflurane, and heparinized. Blood was centrifuged at 1800g for 5 min and the plasma and buffy coat were discarded. Erythrocytes were then washed 4 times in twice their volume of 0.9% saline, with centrifugation for 5 min at 1200g. Packed erythrocytes were then lysed in 10 × their volume of RIPA buffer , and 10 µl of the resultant lysate was separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard protocols.For western blot analysis of erythrocyte PMCA expression, blood was collected from the jugular vein of PMCA4Membranes were blocked in 3% BSA (for PMCA4 detection) or 3% non-fat milk (for PMCA1 detection) for 1 h at room temperature, before refrigerated incubation overnight with primary antibodies against PMCA4 (clone JA9-Abcam) or PMCA1 (clone F-10-Santa Cruz). Proteins were visualized the following day using enhanced chemiluminescence , after incubation with HRP-linked anti-mouse or anti-rabbit secondary antibody  on a ChemiDoc XRS Imaging System (Biorad). Membranes were then incubated with β-actin or GAPDH antibody (abcam) as loading control.ΟC with anti-Ter119 (Ter119—BioLegend), anti-CD44 (IM7—Thermofisher) and anti-CD71 (R17217—Thermofisher), before being washed and resuspended in PBS containing DAPI. Cells were analysed with an ImageStream X Mk II (Amnis) and data was analysed with IDEAS (Amnis).Heparinized blood obtained from the tail vein was surface stained for 25 min at 4 −/− mice on days 7 and 9 of P. chabaudi and P. yoelii infection were uploaded to Image Pro Premier image analysis software. Automated classification of RBCs was performed with a lower size threshold of 3000 pixels  and a roundness threshold of < 1.2 (to exclude overlapping cell populations). A total of 38 fields of view from 3 mice per group per time point was analysed.Images of Giemsa-stained thin blood smears from WT and PMCA4−/− and WT control mice and homogenized through a 70 µm cell sieve to create single cell suspensions (BD Biosciences). RBCs were lysed in the samples by addition of RBC lysing buffer (BD Biosciences), following which cells were washed and resuspended in HBSS containing 2% FCS (FACS buffer). Brains were isolated from mice after intra-cardial perfusion with PBS, chopped into small pieces using scissors, and passed through a 10 ml syringe, before being incubated on a tube roller at room temperature in FACS buffer with Collagenase  (Sigma) for 45 min. The resultant brain cell suspensions were filtered through a 70 µm cell sieve, washed in FACS buffer, and layered on a 30% Percoll gradient and centrifuged at 2000g for 10 min. The supernatant was discarded and the cell pellet collected. RBCs were lysed in the samples by addition of RBC lysing buffer (BD Biosciences) and cells were washed and resuspended in FACS buffer. Absolute cell numbers were determined by microscopy using a haemocytometer and live/dead differentiation was performed using the trypan blue exclusion cell viability assay (Sigma).Spleens were removed from infected and uninfected PMCA4Spleen and brain samples were surface stained for 20 min with anti-mouse CD4 (GK1.5—Thermofisher), anti-mouse CD8a (53–6.7—Thermofisher), anti-mouse CD11a (M17/4—Thermofisher), anti-mouse CD11b (M1/70—BioLegend), anti-mouse CD31 (390—BioLegend), anti-mouse CD45 (30-F11—Thermofisher), anti-mouse CD49d (R1-2—BioLegend), anti-mouse ICAM-1 (YN1/1.7.4—BioLegend), anti-mouse Ly6C (HK1.4—BioLegend) and anti-mouse Ly6G (1A8—BioLegend). Intracellular staining for granzyme B (GB11—BioLegend) was performed for 45 min, after treatment with Foxp3 fixation/permeabilization buffer (Thermofisher). Dead cells were identified and exluded from analysis using LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technologies). Fluoresce minus one controls were used to set gates. Cells were analysed with a BD LSR II (Becton Dickinson) using BD FACSDiva software (Becton Dickinson). Data was analysed with FlowJo (Tree Star Inc.).t test (for parametric data) or the Mann–Whitney U test (for non-parametric data) was used for comparison between two groups. One-way ANOVA, with Tukey post hoc analysis, or the Kruskal–Wallis test, with Dunn post hoc analysis, were used for comparisons between three or more groups, for parametric and non-parametric data, respectively. Survival data was analysed using the Mantel-Cox test. Results were considered as significantly different when p < 0.05.Data was tested for normality using the Shapiro–Wilk test. Unpaired two tailed Pmca4 gene  were analysed [−/− mice, compared with WT control mice  values. Moreover, RBCs of PMCA4−/− mice also had lower mean corpuscular haemoglobin (MCH) values than RBCs from control mice, suggesting that the cells in the PMCA4−/− mice had less haemoglobin per RBC. Other RBC parameters such as RBC total count, haemoglobin level and haematocrit were not altered in PMCA4−/− mice compared with control mice, and total white blood cell count was also not affected by PMCA4 deletion  within the RBC (Ter119+) population between PMCA4−/− and WT mice  observed in the analyses (results not shown). The image stream analysis did show that the mean circularity ratio of mature Ter119+ normocytes and CD71+ reticulocytes was slightly lower in PMCA4−/− mice, suggesting that PMCA4 deficiency affected RBC morphology , and potentially had a small impact on RBC size.Combined, these data indicate that the deletion of PMCA4 had an effect in modulating the haemoglobin content of RBCs  and which causes recrudescing and chronic malarial disease [−/− and WT mice (as shown on day 7 and 9 infection) , in PMCA4−/− mice compared with WT mice , diameter and circularity of RBCs during the course of P. yoelii infection between PMCA4−/− and WT mice  surviving the window period of ECM development (day 6–10)  Fig. B. In agrice Fig. C. This q−/− mice to ECM was associated with alterations in the splenic and/or intracerebral T cell response. Abrogation of PMCA4 did not significantly alter the numbers of activated (antigen-experienced) CD4+ and CD8+ T cells  and intracerebral antigen-experienced CD4+ and CD8+ T cells expressed slightly (but significantly) lower levels of GrB in infected PMCA4−/− mice than in WT littermate mice , was also comparable in infected PMCA4−/− and WT mice on day 7 of infection  ine B Fig. A-F. HoweP. berghei infection, which is a common murine model of cerebral malaria. However, PMCA4 deficiency did not alter the course of non-lethal malaria during P. chabaudi and P. yoelii infections. Moreover, the deletion of PMCA4 did not impact peripheral parasite levels during any of the murine malaria models examined.The key finding of our study is that genetic ablation of PMCA4 expression in mice slightly improved the survival against 2+ levels) appear to be associated with protection against severe malaria [Plasmodium parasites, with P. falciparum parasites being unable to invade dehydrated cells, potentially due to membrane-cytoskeletal alterations precluding correct orientation of merozoites [−/− mice compared with WT mice, and that peripheral parasitaemia levels were not altered in PMCA4−/− mice during any of the investigated murine Plasmodium spp. infections within this study.The PMCA4 SNPs that have been associated with risk to severe malarial disease are believed to control the level of PMCA4 expressed on RBCs . Specifi malaria . PMCA4-d malaria . The hydrozoites , 35. Thu−/− mice, the majority of other analysed RBC properties  were only minorly affected or were unaffected by abrogation of PMCA4 expression. Indeed, whilst the results from the automated blood analyser suggested that mean corpuscular volume of RBCs was lower in PMCA4−/− mice, this was not recapitulated within the image stream analysis. There were also no obvious signs of increased proportions of crenated and dehydrated RBCs, or difference in RBC diameter, perimeter and circulaty in infected PMCA4−/− mice when studying Giemsa-stained thin blood smears. Thus, the data suggests that abrogation of PMCA4 in murine RBCs may not perturb RBC properties to a sufficient level to affect Plasmodium parasite invasion, growth or survival. Indeed, the effect of RBC dehydration on parasite invasion and growth appears to be a graded phenomenon that only becomes significant above a threshold level of dehydration [Plasmodium invasion, growth or survival within RBCs (normocytes or reticulocytes) during murine malaria.Whilst the above analyses did show differences in the mean corpuscular haemoglobin levels in RBCs from PMCA4ydration . The finPlasmodium parasite growth in RBCs. To directly test this question it will be necessary to isolate RBCs from individuals with different PMCA4 SNPs and culture them in vitro with P. falciparum parasites. As intracellular Ca2+ levels dictate many different biophysical properties of RBCs [Plasmodium proteins and cellular deformability, that could influence the development of severe malaria, and which may feature more prominently in human blood-stage malaria than murine malaria. Moreover, in extreme cases, alterations in the morphology of RBCs may lead to increased phagocytosis within the spleen by red pulp macrophages, which clear damaged and old RBCs from circulation.It is important to note that it has yet to be definitively shown in any study that PMCA4 directly affects  of RBCs , the pos2+ level [2+ level. This is in line with the finding in PMCA4−/− mice that the levels of parasitaemia following P. chabaudi and P. yoelii infection were not different between WT and PMCA4−/− mice.Several studies have shown that some genetic conditions involving haemolytic anaemia  are often associated with “leakiness” of the RBC membrane, which lead to the increase in intracellular Ca2+ level . However2+ level  and redu2+ level , but not−/− mice compared with control mice during P. berghei infection, corresponding with the slightly increased resistance of PMCA4−/− mice to ECM. In addition, there was a trend towards lower numbers of T cells in the brains of PMCA4−/− mice during Plasmodium berghei infection, and intracerebral T cells that did accumulate also expressed lower levels of Granzyme B in infected PMCA4−/− mice. Although the IFN-g production by brain accumulating CD8+ T cells was not assessed, it can be anticipated that the level of this cytokine may also have been lower within infected PMCA4−/− mice than in infected control WT mice, given that Granzyme B and IFN-g are sensitive measures of CD8+ T cell pathogenic activity during P. berghei infection [−/− mice compared with control WT mice during P. berghei infection. This suggests that global PMCA4 deletion did not lead to a generalized amelioration of neuroinflammation during P. berghei infection. Thus, further work will be required to assess whether the specific alterations in T cell activity in the brain of infected PMCA4−/− mice, and the associated slight increase in resistance to ECM, was due to cell autonomous effects of PMCA4 deficiency or was caused by other discrete indirect effects within the brain. Indirect effects that may influence CD8+ T cell responses in the brain during P. berghei ANKA infection could potentially include reduced pRBC accumulation and cross presentation of parasite antigen by brain endothelial cells, or alterations in CXCL9 and CXCL10 production, which are critical events in the pathogenesis of ECM [Although the focus of the present study was on the potential role of PMCA4 on RBCs in influencing the course of blood stage malaria, the PMCA isoforms 1 and 4 are ubiquitously expressed in most organs and cell types . Interesnfection . Notablys of ECM –48.−/− mice indicate that although PMCA4 ablation in RBC does not seem to affect Plasmodium life cycle in RBCs, PMCA4 may be involved, at least in part, in the development of cerebral malaria.Studies using PMCA4Additional file 1. Additional figures and tables.


Luciferase activity was examined as iBNP and the iBNP-to-BNP ratio was evaluated. Patient characteristics and clinical parameters were compared, and multivariate regression analysis was performed to determine independent predictors of the iBNP-to-BNP ratio. Female sex and frequencies of atrial fibrillation, hypertension and the use of a calcium channel blocker (CCB) were higher in HFpEF. The iBNP-to-BNP ratio was significantly higher in HFpEF (26.9) than in HFrEF . Multivariate regression analysis identified the existence of HFpEF as an independent predictor of the iBNP-to-BNP ratio after adjusting for all other measurements . Age, hemoglobin, CCB usage and deceleration time were also independent predictors . These results indicate that the elevated BNP in patients with HFpEF is partly due to iBNP from the heart.Brain natriuretic peptide (BNP) levels are increased in both patients with heart failure with preserved (HFpEF) and reduced ejection fraction (HFrEF), but the reasons for this remain unclear. Our purpose was to examine whether serum-induced BNP (iBNP) expression partly contributes to increased BNP in patients with HFpEF. BNP reporter cardiomyocytes from pBNP-luc-KI mice were stimulated with serum from patients with HFpEF or HFrEF ( The number of patients with heart failure with preserved ejection fraction (HFpEF) is increasing, representing a growing burden as a health problem around the world ,2. PrognBrain natriuretic peptide (BNP) belongs to a family of vasoactive peptide hormones with favorable physiological properties . BNP is In this study, 196 patients with HF were recruited . Table 1BNP gene, NPPB (To generate the BNP reporter mice (pBNP-luc-KI mice), the luciferase cDNA was inserted into the initiation site of the ne, NPPB . In vivone, NPPB . Next, wne, NPPB A. Interene, NPPB B.Although BNP levels are well-known to be lower in HFpEF than in HFrEF , we examMany investigators have tried to clarify differences between HFpEF and HFrEF, including predictors of onset , comorbiWe identified the existence of HFpEF as an independent predictor of the iBNP-to-BNP ratio, in addition to age, hemoglobin, use of CCBs, and deceleration time. The finding that age is an independent predictor of the iBNP-to-BNP ratio is interesting. Indeed, elderly individuals are well-known to show higher BNP concentrations even in the absence of cardiac disease . HemogloRecently, ARNI have been used as a first-line therapy for patients with HF. ARNI play an important role in the treatment of HF by raising endogenous natriuretic peptides including BNP . It mighObesity and diabetes mellitus have also been reported to contribute to systemic inflammation, leading to cardiac remodeling and the development of HFpEF ,38. AlthIn summary, we have developed a system to measure iBNP using KI technology. This system showed that the iBNP-to-BNP ratio was significantly higher in HFpEF than in HFrEF, suggesting that iBNP may play an important role in the development of HFpEF and may help clarify the pathophysiological differences between HFpEF and HFrEF.All protocols were approved by the Institutional Animal Care and Use Committee of Mie University (protocol no. 24–25) and RIKEN Kobe Branch .http://www.clst.riken.jp/arg/mutant%20mice%20list.html, accessed on 7 December 2021), the luciferase cDNA was inserted into the initiation site of the mouse BNP gene  A targeting vector was constructed to replace the coding region of exon 1 of NPPB with a DNA fragment containing a luciferase cDNA and loxP-flanked neomycin resistance gene driven by PGK promoter (ryuji@clin.medic.mie-u.ac.jp).To generate the BNP in reporter mice (Accession. No. CDB1373K: promoter . A dipht2/steradian) within a region of interest (ROI). Serial images were collected from the heart in the ventral view for 40 min after luciferin injection in 5-min intervals.Mice received 125 mg/kg of luciferin substrate by intraperitoneal injection. Image acquisition was performed on anesthetized mice receiving isoflurane (2%). Bioluminescence signals were collected using an in vivo imaging system . Light intensity of bioluminescence was recorded as maximum photon counts  by ligation of the left anterior descending coronary artery was studied in 10 to 12-week-old male C57BL/6J mice (SLC), as described previously . AnestheMouse neonatal cardiomyocytes were isolated from the ventricles of 2-day-old pBNP-luc-KI mice and luciferase (luc) knock-in (KI) mice with site-specific integration of the luciferase gene, which is regulated by, and acts as an endogenous promotor of, BNP (pBNP), as described previously . Cells wThe study protocol was approved by the Ethics Committee at Mie University Hospital (reference number: 2893). This study was performed using an opt-out methodology. The opt-out option was presented on the hospital’s website and as a notice in a prominent place at Mie University Hospital.HF was defined using a combination of signs and symptoms, as previously reported . We divi–Whitney test was used to compare baseline characteristics. Multivariate regression analyses were performed to identify factors from among risk factors and laboratory data that were associated with the ratio of iBNP to BNP (iBNP-to-BNP ratio). Values of p < 0.05 were considered significant. Data were processed using SPSS version 25 software .Data are reported as mean ± standard deviation. The chi-squared test or MannThese results indicate that the elevated BNP in patients with HFpEF is partly due to iBNP from the heart.Our recruited patients did not receive angiotensin receptor neprilysin inhibitor (ARNI) or sodium/glucose cotransporter 2 inhibitor (SGLT2i), which are important drugs in the latest guideline , because


African Health Sciences is unique in many ways: it ends the year that might be described using the Queen of England's own words: annus horribilis.1This December 2020, bumper harvest, issue of fake news or rather very poor science: anecdote, case series, poorly designed studies etc. With so much genuine hysteria about such a horrible pandemic there has emerged, inevitably, what WHO calls an infodemic.th alone, AHS has received over 2000 manuscripts double the number we would expect in a year! This has placed enormous pressure on all our volunteer establishment and has inevitably meant increased rejection of manuscripts to the annoyance of our authors. The process of peer review e.t.c, has also slowed down with the expected consternation.It has been a horrible year indeed with record numbers of lives lost to Covid-19; record numbers of papers published on one disease in less than a year: a whooping 750000 papers on Covid-19 alone and still counting. What is disturbing, though, is that most of these papers are not very far from In this issue we purposely have only two papers on Covid_19.In the first one, Emirate scientistsWe do apologize to our stakeholders for the process slow down. We hope that you will enjoy this bumper issue none the less.