V) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs’ structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions’ thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SP Potexvirus, family Alphaflexiviridae  values were calculated taking the mean molecular weight of amino acid residues to be 110. The intrinsic fluorescence spectra were recorded in 1 cm cells, using a FluoroMax spectrofluorometer , at 25°C. Fluorescence was excited at 280 nm and the emission spectra were recorded in the 300–400 nm range.Milli-Q water was used in the case of AltMV VLPs and SPV/VLP were mixed with freshly prepared ThT  according to [AltMV virions, VLPs and SPrding to . The fluV/VLP was carried out in accordance with the manufacturer’s protocol, with some modifications. The initial concentration of the samples before labelling was at least 1 mg/ml. Labelling was performed in 0.1 M sodium carbonate buffer, pH 9. To 1 ml of samples, 50 μl of FITC solution (1 mg/ml) was added and then samples were incubated in the dark, overnight, at 4°C. To remove unbound FITC, the labelled samples were dialysed for 4 h against phosphate-buffered saline (PBS) and for 1 h against Milli-Q water. The 5--fluorescein diacetate  labelling procedure was carried out in accordance with [The procedure of fluorescein isothiocyanate (FITC)  labelling of AltMV virions, VLPs and SPnce with . The resV/VLP were incubated with 10 μg of model antigen, , in water and at room temperature, overnight. Then, the complexes were centrifuged at Microspin FV-2400 for 5 min to remove unbound proteins. The sedimented complexes were resuspended in Milli-Q water and prepared for immunofluorescence microscopy according to a protocol described previously [100 μg of SPeviously . For compost hoc Tukey HSD test. Probability values  of less than 0.05 were considered to be significant. The data are presented as mean ± SD.SP size statistical differences were analysed using one-way ANOVA with a CD spectra were recorded three to five times, averaged and smoothed using the instrument software Pro Data. Two independent experiments were carried out. The data of the secondary structure content are calculated as the mean ± SEM of two experiments.In the case of intrinsic fluorescence and ThT assay, three independent experiments were conducted. Fluorescence maxima are calculated as the mean ± SEM.v [v, an attempt was made to heat virions to 98°С, in various buffers  for 10 sec. At 50°C, only unmodified AltMV virions were detected . Howeverormation . There w or 98°C .V were produced after heating at 94°C, mature PVX SP were detected at 90°C [V need 0.15 М NaCl, while PVX SP form in Milli-Q water or in 0.01 M Tris-HCl, pH 7.8. This hypothesis is consistent with data that suggest that NaCl may increase proteins’ thermal stability [Therefore, the temperatures required for structural transformation are not similar for two flexible filamentous potexviruses (AltMV and PVX). The AltMV IF was observed at a lower temperature (55°C for AltMV) than the PVX (70°C); while mature AltMV SP at 90°C . It is atability . DiffereVLP was also studied. No IF was detected similar to virions with a swollen end. At 40°C, morphologically stable VLPs were observed  with the same concentration, SPous work , we repoV, the following experiment was conducted. AltMV SPV with a concentration of 1 mg/ml were precipitated by low-speed centrifugation, as described in Materials and Methods. The SPV pellet was resuspended in Milli-Q water and was used for RNA isolation (sample 1). Also, RNA was extracted from supernatant (sample 2), from intact SPV preparation which had not been centrifuged (sample 3) and from an equal quantity of untreated AltMV virions (sample 4). No viral RNA was detected in sample 1  after thermal transition to SP ,7. In thnd SPVLP  were proV and SPVLP formation was accompanied by alteration of secondary structure. The content of α-helices in CP in AltMV virions and VLPs was higher than in protein within both types of SP. At the same time, the proportion of β-structure increased after thermal treatment.The analysis of the data obtained confirmed that, similar to SP from TMV and PVX, AltMV SPV and SPVLP)  to 344±2 nm (AltMV SPd SPVLP) . The maxt of TMV  and PVX t of TMV .VLP and SPV . SPV–TM2e and SPVLP–TM2e complexes were prepared by simple mixing with a mass ratio of 10:1 and they were analysed by immunofluorescence microscopy  and 5--fluorescein diacetate. AltMV CP contains eight Lys which could bind FITC and two Cys potentially available to 5--fluorescein diacetate. SPcroscopy . AccordiV and SPVLP. Comparing the fluorescence intensity of labelled virions and VLPs it can be concluded that CP within virions and VLPs possess different surface amino acids composition. Thus, we completed our earlier results [SDS-PAGE electrophoresis, with subsequent visualisation using UV-light , demonst results  obtainedV surface amino acids capable of reacting with FITC and 5--fluorescein diacetate. The same conclusions had been reached for TMV virions and TMV SP labelled by FITC [V and SPVLP are attractive for amino groups’ labelling/conjugation. Herewith, AltMV SPV/VLP is more promising than virions and VLPs for thiol labelling/conjugation.The thermal denaturation of AltMV virions probably led to the appearance on SP by FITC . These dV formation from AltMV virions has been described for the first time. Differences in the thermal remodelling of AltMV virions and VLPs were detected. AltMV virions transformed into SPVvia intermediate forms, as demonstrated for TMV and PVX. AltMV VLPs converted to SPVLP without analogous intermediates. The temperature of the complete structural transition of AltMV virions was 94°C, and for VLPs it was 65°C. According to the biochemical analysis, SPV did not contain viral RNA. Therefore, both SPV/VLP are exclusively protein particles. Differences in the secondary and tertiary structures of CP within untreated virions, VLPs and SPV/VLP, were revealed. The intrinsic fluorescence spectra demonstrate that Trp and Tyr residues are located in the more hydrophilic environment in SPV/VLP. CP within SPV/VLP contained more β-structures and fewer α-helixes than virions and VLPs. This is a reflection of SPV/VLP formation being accompanied by significant changes in secondary and tertiary structures. The possibility of bioconjugation reactions targeted at Lys and Cys amino acid residues in AltMV SPV/VLP has been demonstrated. In this way, AltMV SPV/VLP represent protein platforms with exposed on their surface Lys/Cys amino acids. This makes it possible to apply AltMV SPV/VLP as a base for the formation of functionally active complexes through conjugation with surface reactive amino acid residues or through non-specific adsorption.Thus, the possibility of SPS1 FigA) PBS, pH 7.4; B) Milli-Q; C) 0.01 M Tris-HCl, pH 8.0; D) 0.001 M citrate buffer, pH 4–4.2; E) phosphate buffer, pH 7.4; F) 0.15 M NaCl. Thermal treatment at 98°C for 30 sec. Transmission electron microscopy, staining with 2% uranyl acetate. Bars, 1μm.(TIF)Click here for additional data file.S2 FigV for each concentration. Error bars represent standard deviation. Statistical differences were analysed using one-way ANOVA with a post hoc Tukey HSD Test, **p <0.01.Summary data from four separate samples of SP(TIF)Click here for additional data file.S3 Fig1) Protein molecular weight markers, kDa; 2) 3) AltMV virions; 4) AltMV SPV; 5) AltMV VLPs; 6) AltMV SPVLP. 8–20% SDS-PAGE, staining with Coomassie G-250.(TIF)Click here for additional data file.S4 FigSpectra were recorded at 25°C. The solution details are specified in Materials and Methods. Three repeated experiments were carried out with similar results. The spectra presented in the figure are the visualization of one repetition.(TIF)Click here for additional data file.S5 FigA) Complexes with SPV: 1.–Fluorescence image. 2.–the same image in phase contrast. B) Complexes with SPVLP: 1.–Fluorescence image. 2.–the same image in phase contrast. Immunofluorescence microscopy. Secondary antibodies conjugated with fluorophore Alexa 546.The samples were prepared in the same way as in the experiment see  but the (TIF)Click here for additional data file.S6 FigA) Labelling by FITC. AltMV within the following structures: lane 3 –AltMV virions, lane 4 –AltMV VLPs, lane 5 –AltMV SPV, lane 6 –AltMV SPVLP. Lane 1 –protein molecular weight markers, kDa, lane 2 –unlabelled AltMV virions. 8–20% SDS-PAGE. A1) Staining with Coomassie G-250, A2) visualisation in UV-light. B) Labelling by 5--fluorescein diacetate. AltMV CP included in the following structures: lanes 2,3 –AltMV VLPs, lanes 4,5 –AltMV SPVLP, lanes 6,7 –AltMV virions, lanes 8,9 –AltMV SPV. B1) Staining with Coomassie G-250, B2) visualisation in UV-light.(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Chronic liver disease (CLD) is a global threat to the human population, with manifestations resulting from alcohol-related liver disease (ALD) and non-alcohol fatty liver disease (NAFLD). NAFLD, if not treated, may progress to non-alcoholic steatohepatitis (NASH). Furthermore, inflammation leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Vitexin, a natural flavonoid, has been recently reported for inhibiting NAFLD. It is a lipogenesis inhibitor and activates lipolysis and fatty acid oxidation. In addition, owing to its antioxidant properties, it appeared as a hepatoprotective candidate. However, it exhibits low bioavailability and low efficacy due to its hydrophobic nature. A novel rat model for liver cirrhosis was developed by CCL4/Urethane co-administration. Vitexin encapsulated liposomes were synthesized by the ‘thin-film hydration’ method. Polyethylene glycol (PEG) was coated on liposomes to enhance stability and stealth effect. The diseased rats were then treated with vitexin and PEGylated vitexin liposomes, administered intravenously and orally. Results ascertained the liposomal encapsulation of vitexin and subsequent PEG coating to be a substantial strategy for treating liver cirrhosis through oral drug delivery. Fibrosis delineates the substitution of damaged tissue by creating a collagenous scar . It is fLiver cirrhosis is the 11th leading cause of death worldwide, accounting for 3.5% of all deaths ,8. It haClinically, cirrhosis is considered an end-stage disease that leads to death if liver transplantation is not performed . TreatmeOne of the treatment options for cirrhosis reversal is the administration of antifibrotic agents. Research has been focused on the use of animal models to study the effects of antifibrotic agents on the reversal of cirrhosis . The curVitexin, an 8-Dglucosyl-40, 5, 7-trihydroxy-flavone, has several pharmacological activities such as anticancer, antioxidant, anti-inflammatory, antihypertensive, antiviral, and antidepressant activity ,23,24,25In this study, a novel model of liver cirrhosis is developed to observe the antifibrotic activity of vitexin in Sprague-Dawley Rats. Moreover, polyethylene glycol (PEG) coated vitexin liposomes (VLPs) were prepared, characterized, and realized for vitexin intravenous and oral delivery to treat cirrhosis in rats.DPPC, cholesterol, commercially available vitexin, and PEG (Molecular weight 6000) were purchased from Sigma-Aldrich . Urethane and CCl4 were purchased from Strem Chemicals. Peanut oil (a delivery agent), ethanol (de-contaminant), and 10% neutral formaldehyde buffer (dissolved in PBS) were used. Sprague Dawley female rats were purchased from National Institute of Health (NIH), Islamabad.For the synthesis of liposomes, DPPC and cholesterol were used in 4:1 (percent molar ratio). At first, the lipid was weighed and dissolved in ethanol to form a 100 μM solution. 200 μM solution of vitexin drug was prepared in ethanol from which 500 μL drug solution was taken and mixed in lipid solution. The mixture was sonicated (at 80 MHz) for 40 min. Then 10 mL of water and lipid phase was allowed to be warmed in a water bath individually until the temperature reached 60 °C. The lipid phase was mixed with the water phase and this dispersion mixture was constantly mixed for ten minutes at 90 RPM. This new mixture was again sonicated (for 40 min at 50 MHz) and then allowed rotary evaporation  to remove ethanol. Finally, the unentrapped drug was removed via mini column filtration (0.2 μm size) by centrifugation (at 4500 rpm) for 1 h .The same method that is used for the synthesis of VLPs was implicated for PEG-coated VLPs synthesis with few additional steps. Likewise, 100 μM solutions of lipid and 200 μM of vitexin drug were prepared in ethanol, from which 500 μL drug solution was taken and mixed in lipid solution. Following the sonication, the water phase was added to the lipid phase and a new solution was allowed to rotary evaporation. The next step was to cap the prepared VLPs, for which the mixture was diluted up to 50 mL; then 0.25% PEG was added dropwise, upon continuous stirring, and then allowed to rotary evaporation until 10 mL solution was left behind. The unentrapped drug was removed via mini column filtration by centrifugation (at 4500 rpm) for 1 h .Characterization of VLPs and PEG-VLPs was done to evaluate and analyze their particle size, shape and surface charge, drug encapsulation, release efficiency, and dispersity Index.−1 using Bruker FTIR Spectrophotometer ALPHA II .UV-Vis spectra were measured by using Shimatzu UV-Vis 2800 BMS Scientific Technical Corporation (PVT) spectrophotometer , from 200–450 nm at a resolution of 1 nm. The de-ionized water was used as a reference for UV analysis. The UV spectra of Vitexin drug, blanks LPs, VLPs, and PEG-VLPs were recorded. For FTIR analysis, samples were allowed to air dry and then prepared using compressed KBr discs. FTIR spectra were recorded between 4000–350 cmBoth VLPs and PEG-VLPs were visualized by pouring a 200 μL sample on a coverslip using micropipette. The slide surface was then coated with gold in a sputter coater for 50 s at mA. Images were taken by using VEGA3 LMU Scanning Electron Microscope .Zeta potential (surface charge), size distribution, and dispersity of VLPs and PEG-VLPs were evaluated by Dynamic Light Scattering (DLS) using Malvern Zeta Sizer Ver. 7.12 .To find the congenial concentration of the vitexin for efficient encapsulation within liposomes, different dilutions of the vitexin were made and analyzed by UV spectrophotometer (200–450 nm) to obtain the possible linear standard curve. This standard curve value was further used in calculations to find out the unentrapped vitexin. Samples were centrifuged at 4500 rpm for 1 h, and supernatants were analyzed under UV-Vis. spectrometry to find unentrapped vitexin fractions . After tA total of 3 mL of VLPs and PEG-VLPs solutions were transferred separately into a 15 mL centrifuge tube and allowed for centrifugation for 10 min at 4500 rpm at 25 °C. On the other hand, 3 mL Phosphate buffer saline was added to VLPs and PEG-VLPs solutions. Following centrifugation, the supernatant was allowed for UV spectrophotometer analysis. Same procedure was followed after 1, 2, 4, 6, 12, 24, and 48 h. Analysis was carried out with an empty nanoparticle solution used as a control.60 Sprague-Dawley female rats weighing 85–105 g and ages of 4–6 weeks were used. The rats were kept under a 12-h light and dark cycle in separate cages with accessible water and food. The temperature was set around 27 °C with 60–70% humidity. Chloroform was used to anesthetize the rats to follow histological examination. Handling and caring of rats were centered upon the regulation of good laboratory practice issued by the US FDA (Food and Drug Administration) in 1978.To evaluate the antifibrotic/anti-cirrhotic effects of VLPs and PEG-VLPs, diseased rats were categorized into different groups . Body, lv/v), and their intraperitoneal injections having the dose of 1 mL/kg were given twice a week for the rest of four weeks [Initially, the rats were set free for one week. Normal rats were kept without exposing them to any adverse treatment in the entire procedure while diseased rats were exposed to detrimental chemicals via intraperitoneal injections. Additionally, 2.5% urethane was dissolved in Dimethyl sulfoxide (DMSO) and for the first two weeks, 1 mL/kg dose of urethane was injected intra-peritoneally twice a week. Then, CCl4 was mixed with pure peanut oil  staining was carried out to observe the structural changes of hepatic tissue and hyperplasia of collagen fibers which can be observed in histopathological slides. The pathological grading was based on the criteria of Histological Grading and Staging for Fibrosis by NASH/NAFLD Clinical Research Network scoring system definition and scores , but somVLPs were prepared by the thin-film hydration method. For instance, Dipalmitoyl phosphatidylcholine (DPPC) and cholesterol were used to make a phospholipid bilayer vesicle. DPPC is a phospholipid (and lecithin). It consists of two C16 palmitic acid groups attached to a phosphatidylcholine head-group. The pictorial presentation of the liposomes and structure of vitexin is shown in UV-VIS absorption spectroscopy of vitexin a showed The FTIR spectrum b of cholThe morphology and size of VLPs are visualized through Scanning Electron Microscopy (SEM) a. The imThe mean size of Blank LPs, VLPs, and PEG-VLPs s measured by Zeta sizer were 128 nm, 168 nm, and 458 nm, respectively. The average zeta potential of LPs, VLPs, and PEG-VLPs were −5.73 mV, −9.59 mV, and −0.3 mV, respectively, with a polydispersity index (PDI) of 0.488, 0.24, and 0.498, respectively. The use of PEG-6000 showed a notable increase in the zeta potential of VLPs, suggesting enhanced stability of VLPs.The encapsulation efficiency was found to be 80%, delineating 80% entrapment of vitexin within liposomes.The percentage cumulative drug release (CDR) was measured every two hours until 48 h. The release of vitexin from VLPs, and PEG-VLPs was 72% and 42%, respectively noted up to 48 h. This suggested the sustained release of drug with time from VLPs and PEG-VLPs b. Vitexi, urethane and peanut oil was used to induce cirrhosis. The histopathological, serological analysis, body and liver weight analysis, and ascites observations confirmed the successful induction of cirrhosis in rats.The combination of CCL4The histopathology results confirmed the induction of cirrhosis after five weeks. The scoring was done to study the stages of liver cirrhosis . In the The kidneys and spleen also presented the abnormalities associated with hepatotoxicity and cirrhosis .p = 0.00001) (p = 0.00061) (p = 0.00345) (p = 0.00013)  values obtained from the blood of the normal rats and the rats treated with CCl4 and urethane. This difference increased with the progression of weeks as noticed from Alkaline Phosphatase (ALP) (0.00001) a, Total 0.00061) b, Aspart0.00345) c, and Al0.00013) d, being p = 0.05401) and liver weight (p = 0.00011) significantly reduced in the sixth week of cirrhosis induction  and oral gavage (OG)), VLPs (IV), and PEG-VLPs (OG)). The histopathological analysis, serological analysis, body and liver weight, and ascites observations showed the effectiveness of vitexin and PEG-VLPs in treating the cirrhosis-induced rat model by comparing these results with that of the normal rats.The liver, renal, and spleen histopathological analyses are provided in In histopathology of renal tissues of vitexin IV treated group, kidneys’ glomeruli showed mesangial proliferation, 25–50% of cortical subcapsular tubules showed fatty change, necrosis, and vacuolar degeneration, 30% of medullary tubules also showed vacuolar degeneration, and blood vessels were congested. In the vitexin OG treated group, in kidney >20% of glomeruli were shrunken, blood vessels were congested, 20% degeneration in the medullary tubule, and 35% cortical damaged tubules (ATN) was observed. In VLPs IV treated group, renal histology showed the hypercellularity of glomeruli, atrophicity of glomeruli decreased , <20% of tubules showed necrosis and degeneration, no cellular casts were observed, and regenerating tubules were seen. In the PEG-VLPs OG treated group, glomeruli had <10% trophicity, no cellular casts were seen, regenerating tubules were observed, and there was a lower extent of necrotic cells .In spleen tissue histology of the vitexin, IV treated group, hyperplasia of white pulp, congestion, and hyperplasia of red pulp were observed. In the vitexin O.G treated group, germinal centers were prominent, and there was light fibrosis of red pulp but still decreased follicles and congestion of red pulp was seen. In VLPs IV treated group, follicles of variable sizes and increased in number with 50% of germinal centers were prominent. In PEG-VLPs treated group, there was no congestion and hyalinization, but still hyperplasia of red pulp was observed .p = 0.00116, T.B.; p = 0.00808, ALP; p = 0.000059, and ALT; p = 0.000399) . Only the difference in the values of ALP showed that vitexin administered by OG proves to be more effective. In contrast, rest of the LFTs do not specify any considerable difference. Similarly, the difference in the values obtained of ALP (p = 0.000265) from the blood of rats administered intravenously with vitexin treated VLPs and orally with PEG-VLPs suggests that PEG-VLPs are the superior choice for the treatment of liver cirrhosis. Whereas AST (p = 0.32742), T.B. (p = 0.11078) and ALT (p = 0.540678) values did not alter much in the blood of rats administered intravenously with VLPs and orally with PEG-VLPs.The serological indices noticed after performing LFTs showed a significant difference between normal/control and diseased rats  . This inp = 0.00075 for body and p = 0.18252 for liver), indicating the successful development of liver cirrhosis (p = 0.32007 for body and p = 0.00449 for liver), being of comparatively higher value in case of the oral administration, suggesting that vitexin administered by OG is more effective against liver cirrhosis. Weight reduction was seen the lowest in the group orally administered with PEG-VLPs and was also comparatively better than that of the rats intravenously administered with VLPs (p = 0.64807 for body weight and p = 0.02878 for liver weight).Body and liver weight analysis showed a significant decrease from that of the normal rats compared with the diseased rats  . HoweverRecently, vitexin was established to alleviate the non-alcoholic fatty liver disease via activation of AMP-activated protein kinase (AMPK) that resulted in lipolysis and inhibition of lipogenesis . HoweverThe preset research also reported a novel liver cirrhosis model by coadministration of CCL4/urethane. CCL4 is most commonly used hepatotoxin in cirrhosis induction in rodents. It mimics human chronic disease linked with toxic damage. The hepatic biotransformation of CCL4 produces trichloromethyl radical that induces several free radicals and lipid peroxidation reactions that contributes to an excruciating phase reaction characterized by the necrosis of centrilobular hepatocytes . FurtherAfter successful induction of cirrhosis, the antifibrotic effects of vitexin, VLPs and PEG-VLPs were analyzed to validate the model. The histological, serological, body, and liver weight results exhibited the significant reversibility of collagenous scars to normal hepatic tissue paradigm by using the VLPs and PEG-VLPs contrary to the blank vitexin. Fatty changes in the kidney along with tubular necrosis were noticed that perpetuated from mild to severe over the period of five weeks. At the same time, lymphoid follicle formation, hyaline deposition, and subsequent hyperplasia of red pulp in spleen were indicators of successful cirrhosis model development by the end of sixth week.Following treatment of the diseased rats, the histological, serological, body and liver weight results depicted significant improvement in group treated with PEG-VLPs. Apart from that, VLPs treated group also showed better results up to some extent. Thus, for the reversibility of collagenous scars to normal hepatic tissue, the use of the encapsulated vitexin drug is better choice, contrary to blank vitexin. Hence, liposomes encapsulation along with PEG coating is proved as the most effective stratagem in enhancing the bioavailability and pharmacokinetic behavior of vitexin drug. It has also proven the anti-fibrotic activity of considerable therapeutics against liver cirrhosis. In addition to the route of administration for blank drug, the intravenous route has showed better results, although further research is required to study the effects of oral administration. As such, VLP and PEG-VLPs are found to be effective in treatment of liver cirrhosis and are potential candidates for NAFLD/NASH disease management. Future studies are required to study gene/protein expression analysis of inflammatory biomarkers such as interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and monocyte chemoattractant protein 1 (MCP-1) to understand the mechanism of action of cirrhosis reversal by vitexin in detail.“Vitexin Nano formulation for treatment of advance liver disease” is submitted to Intellectual Property Office of National University of Sciences and Technology Islamabad for National Patent.


To investigate if visuomotor coordination of hand movements is impaired in patients with normal pressure hydrocephalus (NPH) identified by dedicated testing procedures.R-device before and after a spinal tap of 40–50 ml CSF. Statistical analyses were carried out with repeated measures ANOVA and non-parametric correlation analyses.Forty-seven patients admitted for diagnostic workup for suspected NPH were studied prospectively with MRI, testing of cognitive and motor functions, lumbar puncture, and visuomotor coordination of hand movements using the PABLOn = 18). In the patients with NPH the deficient arm control was improved after the spinal tap in proportion to the improvement of gait. There was no improvement of cognitive and motor functions in the patients not affected by NPH, while the patients with possible NPH (n = 15) showed intermediate deficit and improvement patterns.Fourteen patients were found to suffer from ideopathic NPH. They were severely impaired in visuomotor control of intermittent arm movements in comparison to patients who were found not to be affected by NPH (Interpretation: Our data underline the importance of a multiparametric assessment of NPH and provide evidence for a motor control deficit in idiopathic NPH involving leg and arm movements. It is suggested that this motor control deficit resulted from an affection of the output tracts from the supplementary motor area in the periventricular vicinity. Normal-pressure hydrocephalus (NPH) established as a neurological syndrome in the pre-neuroimaging era  is charaIn particular, we also applied a quantitative investigator-independent test to explore the hypothesis that NPH does not only affect urine continence and cognitive and gait functions but also functioning of the arm and hand. Specifically, we were interested to assess visuomotor hand coordination using quantitative measures of hand-arm functions that are performed in daily life. For this purpose, we studied hand movement control of items on a computer screen which is a common and widely used task in the present time. We will show that visuomotor coordination of manual actions is impaired in NPH suggesting that these patients suffer from a more general motor deficit than has been appreciated so far.Forty-seven patients of both sexes admitted for diagnostic work-up of putative NPH between August 2015 and June 2017 who had agreed to participate in this experimental investigation were prospectively included into this study. The patients suffered from a recently insidiously progressing impairment of gait and cognitive abilities as well as urine incontinence and have communicating internal hydrocephalus on cranial MRI. According to the International Guidelines the ventricular enlargement was associated with a patent Sylvian aqueduct and absence of a macroscopic obstruction of CSF flow, lack of cortical atrophy, presence of periventricular water content, and an increased callosal angel in the coronal plane , 13. PatThe neuropsychological tests comprised (I) the clock drawing test  to assesGait and mobility were assessed using a repertoire of dedicated tests as detailed previously . These t® -AMYLOID (1–42), INNOTEST® hTAU Ag, INNOTEST® PHOSPHO TAU (181P). Neuron-specific enolase and S100B were measured using the fully automated commercially available chemoluminescence immunoassays LIAISON® S100 and LIAISON® NSE .CSF samples were obtained by lumbar puncture after informed consent and processed as described elsewhere . In shorR-device as detailed previously [Hand movements were assessed with the so-called PABLOeviously . In shorballoon task the scenario displayed mountains, houses, trees and clouds and a hot air balloon which moved with constant speed on screen from the left to the right for 2 min. The balloon had to be steered by a handle involving rotating hand movements. Clockwise hand movements made the balloon going up and anti-clockwise hand movements made the balloon go down on screen. The patients’ task was to steer the balloon such that it would not bump into a mountain or cloud. When the subject failed in performing this highly predictable task and the balloon bumped into the obstacles, the movement speed of the balloon was decreased for a few seconds. There were 10 levels of task difficulty given by an increasing travel speed of the balloon.In the collecting apple task, there were three apple trees with green apples .R-device were entered as raw values. A repeated-measures ANOVA with post-hoc paired t tests of the changes was calculated. T values with alpha-corrections for multiple comparisons were calculated and converted to p-values. Significant change was defined to exceed p < 0.05. Also non-parametric correlation analyses using Spearman Rank correlations (SRC) were calculated. Significant SRC had to exceed a p < 0.05.On the individual level of each patient an improvement of the neuropsychological performance and of the Demmi index after the tap-test was determined for each test as a categorical change in the age, gender and education related mean reference values in comparison to the pre-test values. Furthermore, an improvement in the 10MWT of > 10% was set as a cutoff for improvement (13). In addition, the data were entered into a spreadsheet of SPSS  for group statistics. The values of gait performance and of the PABLOEach patient could perform the neuropsychological test battery and the gait assessment tests as summarized in Table p < 0.05) but improved in this test after the spinal tap (p < 0.02). In addition, the majority of the patients with NPH improved in neuropsychological testing, while some improvement was found only in a small proportion of patients in the other two groups  area.


Electroconvulsive therapy (ECT) is the most effective treatment for severe late-life depression (LLD), and several hypotheses on the precise working mechanism have been proposed. Preclinical evidence suggests that ECT induces changes in neurotrophin and inflammatory signaling and that these neurotrophic and inflammatory systems affect each other. We examine the relation, interaction, and ratio between the neurotrophic brain-derived neurotrophic factor (BDNF) and the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α), and depression severity during ECT.In this naturalistic longitudinal study, linear mixed models were used to analyze the relation between BDNF, IL-6, TNF-α, and depression severity  in 99 patients with severe LLD before ECT (T0), three weeks after the first ECT (T1), and one week after the last ECT (T2).p = .020) at all time points. With higher levels of TNF-α, the relation between BDNF and MADRS becomes more negative. Furthermore, a higher ratio of TNF-α/BDNF was associated with a higher score on the MADRS (p = .007).No significant association was found between BDNF, IL-6 and TNF-α, and MADRS scores at any time point. However, a significant interaction between TNF-α and BDNF in relation to MADRS was established (A possible explanation for the absence of a significant coevolution between the proinflammatory cytokines and BDNF could be that the study design was unable to determine parameters shortly after ECT sessions. However, the TNF-α/BDNF ratio was positively associated with depression severity, and the association of BDNF-level and depression severity depended on the level of TNF-α. This suggests that the interaction and balance between neurotrophin and immune signaling, specifically BDNF and TNF-α, could be relevant in LLD. This could be a focus in future research regarding treatment and the working mechanism of ECT. •IL-6 and TNF-α, BDNF, and depression severity were not significantly associated.•The TNF-α/BDNF ratio is positively associated with depression severity.•The association of BDNF-levels and depression severity depends on the level of TNF-α.•Interplay between neurotrophin and immune signaling could be relevant in LLD. SeveralMajor depression disorder (MDD) could be conceptualized as stress-elicited neuronal micro injury resulting in behavioral disturbances . As a reHowever, severe MDD could lead to a prolonged and detrimental state of inflammation. Hence, pathological inflammation is differentiated from advantageous inflammation by the intensity and timing of its appearance . RecoverPreviously, we found that some degree of proinflammation  and low 22.1In total, 110 older adults with a diagnosis of severe MDD according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria participated . All pat2.2Demographic and clinical variables were obtained by means of a semi-structured interview. To determine the depressive symptom severity, the present study assessed the Montgomery Åsberg Depression Rating Scale (MADRS) on T0, T1, and T2 . The ove2.3ECT was administered twice a week with a constant-current brief-pulse (0.5–1.0 ms) device: Thymatron System IV . Concerning the administration of ECT, Dutch guidelines and standards were maintained . Anesthe2.4g), and assayed instantly. Serum samples containing either low or high cytokine levels were included in each plate run to control quality. BDNF levels were also determined by using the separated serum from blood samples. To accurately increase the detectable BDNF in a dilution-dependent way undiluted serum was acid treated. Greiner Bio-One high affinity 96-well plates were used. Serum samples were diluted 100 times, and the resulting absorbance was read in duplicate using a Bio-Rad  Benchmark microplate reader at 450 nm. For IL-6, the lower limit of detection was 0.006 pg/mL and the lower limit of quantification was 0.011 pg/mL. For TNF-α, this was 0.011 pg/mL and 0.051 pg/mL, respectively. No patient had BDNF values that were below the reliable detection threshold of 1.56 ng/mL. The serum levels were determined in the University Medical Center of Maastricht using the Emax Immuno Assay System from Promega according to the manufacturer's protocol .Blood samples were taken at the three time points mentioned above  to determine IL-6, TNF-α and BDNF cytokine levels. Between 0730 and 0930 h, 50 mL of blood was collected and withdrawn in vacuum tubes after an overnight fast. Serum was instantly separated and stored at −85 °C until being assayed in seven to 43 months after collection. Using the Single Molecule Array (Simoa) technology and the Simoa HD-1 Analyzer, the IL-6 and TNF-α serum levels were determined with a three-step multiplex digital immunoassay, the Simoa Human Cytokine Three-Plex A assay kit . For IL-6, the intra-assay reproducibility was 4.3%, and the inter-assay reproducibility, for which the coefficient of variation was based on data from ten runs, was 5.9%. For TNF-α, this was 4.2% and 5.3%, respectively. Upon thawing, the samples were mixed by inverting, spun at 4 °C , and the data within this variable contained three outliers . These three outliers were not included in the analyses, since these values were highly deviant (more than five to 14 times the standard deviation from the mean) and a result that is merely based on these outliers is less valuable. Moreover, to avoid bias as a result of inflammatory disease and acute infection or treatment such extreme outliers were excluded. Residuals of all analyses were checked to be normally distributed. TNF-α values ranged from 0.0 to 19.7 pg/mL , and BDNF values ranged from 4.03 to 35.6 ng/mL . To investigate the relation between IL-6, TNF-α, BDNF, and MADRS, multilevel or linear mixed models (LMM) were estimated with repeated measurements (level 1) being nested within patients (level 2). LMMs were chosen because they: i) use all available data, ii) can properly account for correlation between repeated measurement on the same subject; iii) allow multivariable analysis; and iv) can handle missing data adequately (p ≥ .050), it was removed from the model to interpret the main effect. Model selection was based on likelihood ratio tests and information criteria.Data were analyzed using the software “Statistical Package for the Social Sciences” . IL-6 values ranged from 0.0 to 145.9 pg/mL  with 17 patients aged 55–64, 36 patients aged 65–74, 40 patients aged 75–84, and six patients aged 85–94. 66.7% of patients were female and the mean administered number of ECT sessions per patient was 12 (SD = 5.53). During treatment course, 31.3% of patients switched to bilateral ECT due to inadequate response with right unilateral ECT. Response was seen in 83.9% of patients and 71.1% achieved remission. The 99 patients in our study sample were included in all linear mixed model analyses executed. The following covariates were significant in analyses where MADRS was the dependent variable: age, time points, switching from unilateral to bilateral electrode placement and total number of received ECT sessions. MADRS scores decreased significantly over time and a lower MADRS score was associated with a higher age. Also, at T1 and T2 a higher MADRS score was associated with a higher total number of received ECT sessions. Patients switching from unilateral to bilateral electrode placement had a significantly higher MADRS score at T1 and T2. The covariate site was significant in the analyses with BDNF and ratio IL-6/BDNF as dependent variables. Patients from Amsterdam had a significantly higher BDNF-levels, F = 38.6, p < .001, and lower ratios IL-6/BDNF, F = 5.07, p = .027, than patients from Leuven. A higher age was also associated with a higher ratio IL-6/BDNF, F = 7.50, p = .007. All covariates not mentioned were not significantly associated with the dependent variable in other analyses. Remission from MDD  and medication use were not significantly associated with dependent variables.Clinical characteristic are shown in 3.2p = .177, over the course of ECT, nor between TNF-α and BDNF, F = 1.04, p = .309, and these associations were not significantly different in remitters than in non-remitters. The next LMMs disclosed no significant association between IL-6 and MADRS, F = 0.95, p = .331, BDNF and MADRS, F = 0.26, p = .611, and TNF-α and MADRS, F = 2.35, p = .127. No interaction between IL-6 and BDNF was found, but the interaction between TNF-α and BDNF was significant, F = 5.55, p = .020. Thus, the effect of BDNF on MADRS depends on the level of TNF-α. With higher levels of TNF-α the relation between BDNF and MADRS becomes more negative (lower BDNF levels with higher MADRS scores), while with lower levels of TNF-α the relation between BDNF and MADRS disappears.The evolution of IL-6, TNF-α, BDNF, and MADRS over the course of ECT is shown in 3.3p = .076 and F = 1.35, p = .261, respectively  = 2.65, ectively . Moreove4In the present study IL-6, TNF-α, BDNF, and depression severity before, during, and after ECT were examined in LLD. No significant association between IL-6, TNF-α, BDNF, and depression severity was established before, during and after ECT. A significant association between the ratio of TNF-α/BDNF and depression severity was found: the higher the TNF-α/BDNF ratio, the greater the depression severity. Interestingly, no significant change in the proinflammatory cytokine/neurotrophin ratios over the course of ECT was found despite the significant decline of depression severity over time. However, a significant interaction between TNF-α and BDNF was demonstrated: the association of BDNF-level and depression severity depends on the TNF-α level.4.1No significant coevolution was found between the proinflammatory cytokines and BDNF. This finding contradicts research results that show a positive association between the proinflammatory cytokines and BDNF in various study samples , possibl4.2A recent meta-analysis found that during ECT treatment there is an initial pro-inflammatory response with increased IL-6 levels eventually followed by decreased IL-6 and TNF-α levels. A possible interpretation given by Gay et al. is that increased IL-6 levels in response to ECT induce neuroplastic change and the eventual decrease in IL-6 and TNF-α levels could be related to recovery of depression or to an anti-inflammatory response of ECT. However, in the present study no significant coevolution was found between IL-6, TNF-α, BDNF, and depression severity. Previously, data from the MODECT study indicated that LLD patients with lower levels of BDNF are more likely to achieve remission with ECT . Nonethe4.3Although no significant association is found between the ratio IL-6/BDNF and depression severity, a significant association between the TNF-α/BDNF and depression severity is established. The latter suggests that the proinflammatory cytokine/neurotrophin balance plays an important role in brain diseases . The preTo our knowledge this is the first study that used the proinflammatory cytokine/BDNF ratios in relation to depression severity during and following ECT. The balance between immune and neurotrophin signaling may be relevant in LLD. The present study was designed to examine the relation between two proinflammatory cytokines, BDNF, and depression severity, and despite the lack of association, the interaction and balance between TNF-α and BDNF in relation with depression severity could underlie a broader psycho-neuroendocrine mechanism of action.4.4The results of the present study should be interpreted in the light of its limitations. On the basis of the present findings, it may not be possible to demonstrate a relation between cytokine and BDNF levels induced by ECT because the study design was incompetent to determine cytokine and BDNF levels shortly after ECT sessions. Additionally, the present study examined LLD and future research should include MDD patients of all ages. Furthermore, differentiating between remitters and non-remitters was more difficult in the present study as relatively few patients did not remit and even less did not respond to ECT at all. Despite its limitations, with a sample of 99 patients the present study is fairly large for an ECT study. Furthermore, a strength of the present study is the careful evaluation and diagnosis of patients by a psychiatrist with establishment by the Mini International Neuropsychiatric Interview. Due to the exclusion of patients with bipolar or schizoaffective disorder the effect of ECT on unipolar depression could be more clearly considered.5The theory that ECT promotes a targeted elevation of the proinflammatory cytokine levels and thereby induce a neurogenic process via BDNF leading to clinical response in LLD could not be supported, since no significant coevolution between the proinflammatory cytokines, BDNF, and depression severity was found. However, sampling closer to ECT sessions is advised to study the more acute effect of ECT and is recommended for further studies to increase our understanding of the neurobiology of ECT.Nevertheless, our findings that the TNF-α/BDNF ratio was positively associated with depression severity, and that the association of BDNF-levels and depression severity depended on the level of TNF-α suggest that the interaction and balance between neurotrophin and immune signaling, specifically BDNF and TNF-α, could be relevant in LLD. This could be a focus in future research regarding LLD treatment and the mechanism of action of ECT.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


The concepts brought by Industry 4.0 have been explored and gradually applied.The cybersecurity impacts on the progress of Industry 4.0 implementations and their interactions with other technologies require constant surveillance, and it is important to forecast cybersecurity-related challenges and trends to prevent and mitigate these impacts. The contributions of this paper are as follows: (1) it presents the results of a systematic review of industry 4.0 regarding attacks, vulnerabilities and defense strategies, (2) it details and classifies the attacks, vulnerabilities and defenses mechanisms, and (3) it presents a discussion of recent challenges and trends regarding cybersecurity-related areas for Industry 4.0. From the systematic review, regarding the attacks, the results show that most attacks are carried out on the network layer, where dos-related and mitm attacks are the most prevalent ones. Regarding vulnerabilities, security flaws in services and source code, and incorrect validations in authentication procedures are highlighted. These are vulnerabilities that can be exploited by dos attacks and buffer overflows in industrial devices and networks. Regarding defense strategies, Blockchain is presented as one of the most relevant technologies under study in terms of defense mechanisms, thanks to its ability to be used in a variety of solutions, from Intrusion Detection Systems to the prevention of Distributed dos attacks, and most defense strategies are presented as an after-attack solution or prevention, in the sense that the defense mechanisms are only placed or thought, only after the harm has been done, and not as a mitigation strategy to prevent the cyberattack. Concerning challenges and trends, the review shows that digital sovereignty, cyber sovereignty, and data sovereignty are recent topics being explored by researchers within the Industry 4.0 scope, and GAIA-X and International Data Spaces are recent initiatives regarding data sovereignty. A discussion of trends is provided, and future challenges are pointed out. Industry 4.0, or the fourth industrial revolution, advocates the automation of traditional practices of manufacturing and industrialization , with thRecent technologies have been integrated within Industry 4.0 implementations, bringing new challenges in the cybersecurity area . Recent With the increase of devices connected to Industry 4.0-enabled networks, the surface of attack also expands. Malicious actors may find in any smart device an open door to exploit new vulnerabilities and perform attacks on them or on their infrastructure, with the intent of impacting financially a company or industry . When coThe contributions of this paper can be divided into three. First, a general systematic review of current cybersecurity attacks, vulnerabilities and defenses in Industry 4.0 and 5.0 scenarios is presented. Second, a detailed analysis and categorization regarding attacks, vulnerabilities and defenses of selected studies is presented. Third, a discussion is presented of recent challenges and trends regarding these areas on Industry 4.0 and Cybersecurity. This review is divided into three steps: (1) General Review, (2) Abstracts Review, and (3) Selected Papers Review.This systematic review allows the identification of the most common attacks, vulnerabilities, and defense strategies. Additionally, a set of challenges and trends regarding Industry 4.0 are highlighted as an effort to enhance the detection and prediction of new vulnerabilities or zero-day attacks, and creating the necessary defense mechanisms to protect industry data. Digital, Cyber and Data Sovereignty concepts are discussed since challenges emerge regarding data sharing and ownership, and recent initiatives such as ids  and GAIAThe remainder of this document is organized as follows: This review around the cybersecurity-related topics in Industry 4.0 intends to overview types of attacks, vulnerabilities and defense strategies. Additionally, this study aims to identify whether topics such as data sovereignty, digital sovereignty and cyber sovereignty are current trends for cybersecurity in the context of Industry 4.0, due to the appearance of the ids and GAIA-X. Thus, a general systematic review was carried out, inspired by the methodology in , adaptedRQ1—What is the progress of the cybersecurity area for Industry 4.0, in number of papers related to vulnerabilities, defense and attacks topics?RQ2—What is the progress, in number of papers for the intersections of the topics of vulnerabilities, attacks and defense mechanisms for Industry 4.0?RQ3—What is the progress of the number of papers for challenges and trends related to data sovereignty, digital sovereignty and cyber sovereignty areas for Industry 4.0?For the current study, we assume that the published paper progress over time (from 2014 to 2021) can be a possible approach to overview the cybersecurity-related topics and infer current trends and challenges. Thus, the following Research Questions (RQ) were formulated:After defining the research questions, search engines were chosen. For this study, we selected the ACM Digital Library, Scopus and IEEExplore databases to receive the queries as input and to provide quantitative results of the number of papers.The keywords used to perform the search queries were defined to include the results of Industry 4.0 and Industry 5.0 and to gather all recent matches regarding attacks, vulnerabilities, defenses, and sovereignty-related terms. The primary and secondary keywords used are presented in In After the first search queries 1640 papers were selected, and duplicates were removed. For the remaining 855 papers, the StArt tool , a suppoA detailed review was performed in three colored steps in In this section, the results of the general review of search queries performed are presented. The number of papers referring to vulnerabilities, defenses, attacks, or the intersection of these three main topics is presented.from 2014 to 2015 the number of papers regarding vulnerabilities where none;from 2014 to 2015 only one paper was published regarding defense strategies or mechanisms;the number of papers regarding attacks was constant until 2015, when it increased until now;papers about defense had a strong increase in number, per year, from all subjects analyzed, but the forecast for 2021 is to maintain, approximately, the previous year’s number;the subject of attack presented the greatest number of papers, with 97 papers in 2020, and an estimate of 111 papers for 2021;the number of papers addressing vulnerabilities, despite growing over the years, has reduced compared to other subjects;the biggest year of growth in papers published was 2019, with a median factor of 2.3 times.the number of papers addressing attacks is the largest, followed by the number of papers including defense mechanisms.the number of papers talking about attacks has been growing significantlythe number of papers talking about vulnerabilities has grown over the years, despite being small, compared to attacks and defensesthe number of papers for both topics is twenty five (35)from 2014 to 2016.between 2017 and 2021 it is possible to identify that the number of papers increase, for all topics, except for the topic of V&D, in which there is a small increase.from 2014 to 2017 for the categories of data, digital and cyber sovereignty, no papers were found.in 2018, the number of papers found was only 1 for the category of cyber sovereignty, which did not have papers found again until 2021 (projection).in 2019, no papers were found for the three categories.for digital sovereignty papers were found only in 2020.for data sovereignty, from 2014 to 2019 the number of papers is zero, but an increase is seen in 2020 and 2021 (projection).Since the queries were extended to included recent areas and possible trends, the results of search queries including data, digital and cyber sovereignty were processed and analyzed. These results are presented in From the 11 papers referring to data, digital or cyber sovereignty, two of them focus on particular initiatives, namely the GAIA-X and the ids. In  the authRQ1—What is the progress of the cybersecurity area for Industry 4.0, in number of papers, relative to vulnerabilities, defense and attacks topics? Answer — The results obtained show that the number of papers for the three topics  have increase from 2014 to 2020. In the current year of 2021, the projection is that the number will be greater for Attacks and Vulnerabilities topics, and the projection for the Defense topic is to be similar to the last year.RQ2—What is the progress, in number of papers for the intersections of the topics of vulnerabilities, attacks and defense mechanisms for Industry 4.0? Answer — Regarding the intersection topics, a strong increase of papers was verified regarding V&A, followed by D&A, and, with a slight increase, the V&D.RQ3—What is the progress of the number of papers for challenges and trends related to data sovereignty, digital sovereignty and cyber sovereignty areas for Industry 4.0? Answer — With the results obtained, it is concluded that digital, cyber and data sovereignty are relatively recent topics that presumably will grow over the years, particularly data sovereignty. Additionally, GAIA-X and ids are initiatives taking shape and intend to tackle the data sovereignty-related challenges.Given all the results regarding the General Review, the research questions formulated can be answered as follows:In this section, a review of the abstracts is presented. The papers with a score greater than or equal to 100 were selected and examined.the number of papers referring to defense mechanisms has grown in recent years, and reaches 24 for 2021 (forecast).there are no papers matching V&D in any of the years in review.between 2014 and 2016, no papers were found.between 2017 and 2021, the number of papers increases for all topics, with the exception of V&D, which remains null.there are no papers that address vulnerabilities and defenses simultaneouslythe largest group of papers have scores from 100 to 130the highest-scoring papers match the three topics: attacks, defenses and vulnerabilities and addresses a risk-assessment of cyber-attacks and defense strategies for Industry 4.0. This paper also reviews the most common cybersecurity vulnerabilities and defense strategies regarding Industry 4.0, in corporate and end-user dimensions.In In this section, a more specific analysis is performed regarding the 22 papers selected after the full text analysis step of the systematic review. In A finer analysis regarding the three main topics was performed and, following a similar approach of the previous works in ,72, a seIn this section, 14 relevant papers were found. As shown in NetworkNetwork attacks are commonly designed to impact a network’s performance. To impact the performance, a malicious actor can perform a dos ,63,64,76MalwareMalware is intended to cause damage, steal or modify information in a target computer. This type of attack can be presented in many shapes or forms, such as a virus ,129, whiWeb ApplicationTo access sensitive data, web application attacks can lead to compromises in a company’s network. Metadata spoofing and sql Injection are types of attacks that threaten security in iiot. Metadata spoofing is when an attacker modifies a database and causes its integrity to be compromised . When thSystemAttacks targeting the ics have increased by 110% since 2016 . For IndDevicesDevices for Industry 4.0, such as sensors, robots and industrial machines, can be attacked by various types of attacks. Physics, measurement injection, side channel and time delay are the most common attacks. Physical attack is when an untrusted worker gains physical access and makes unwanted modifications to devices. Measurement injection is when false data are injected into the sensors. In a side-channel attack, the attacker can gather sensitive information from the device by measuring side-channel information. A time delay attack can disturb the stability of all the industrial control system, by adding extra time delays into measurements and control commands .Social EngineeringAn attacker using social engineering often uses his abilities to convince the user ,129. In Regarding vulnerabilities, seven (7) relevant papers were found. The vulnerabilities studied were divided into 4 categories Web Application, Devices, Network, and Authentication, as presented in Web ApplicationVulnerabilities related to web services and applications are usually associated with coding errors that enable destructive or non-destructive attacks. Vulnerabilities that allow malicious users to execute unwanted scripts , such asDevicesWith regards to iiot devices, such as plc, cameras, smart-routers, smart-meters from various vendors, vulnerabilities were found in the software and firmware of the devices. These are Buffer Overflow, Infinite Loop, uaf, Heap Overflow and ddos vulnerabilities . Buffer NetworkNetwork vulnerabilities are directly related to attacks to devices and services . AccordiAuthenticationAccording to  improperRegarding defense mechanisms, 8 relevant papers were found. The defense mechanisms studied were divided into the following 6 categories, Application, Devices, Network, Social Engineering, Policy, and System, as shown in Application—Application defense can be seen as developing custom defense mechanisms to improve overall safety of a product. Various methods can be selected to achieve a certain desired result. The authors in [thors in  propose thors in  the auththors in . In termthors in , a frameDevices—All devices and software used in organizations should be configured safely, and controlled changes should be ensured [VulHunter, which aims to discover unknown vulnerabilities in the analysis based on the patch of known vulnerabilities. To secure network devices, the authors in [ ensured . In Ref. ensured , the autthors in  address thors in  the auththors in  ai is seNetwork—Traffic between networks with different security levels should be restricted and monitored [onitored . With thonitored  propose onitored  aim to ionitored , the autonitored , in whicSocial Engineering—Defense strategies for social engineering can include collaborators training to recognize sensitive information requests  or a redirection to false web sites that intend to capture this data outside the scope of the corporation. For the authors in [Truecaller or Dialer Software is seen as a method of defense against phishing attacks. According to [thors in , the userding to , emails,Policy—In Ref. [—In Ref. , authors—In Ref.  and ISO/—In Ref.  standard—In Ref. ,129 mustSystem—According to [rding to , Blockchrding to , the autRegarding attacks: the number of papers was constant until 2015, and has been growing significantly since. Papers addressing attacks are the highest in number, followed by the number of papers including defense mechanisms;Regarding defenses: the number of papers had the highest increase in number, per year, from all subjects analyzed, but the forecast for 2021 is to maintain, approximately, the previous year’s number.Regarding vulnerabilities: the number of papers addressing vulnerabilities, despite growing over the years, has reduced compared to attack and defenses topics.Regarding the correlation between topics , between 2017 and 2021 it is possible to identify that for all topics, the number of papers had a strong increase, except for the topic of V&D, which had a small increase.As a result of the systematic review, it is possible to identify the most common types of attacks, vulnerabilities and defense mechanisms for Industry 4.0, and check their progress in recent years. The main highlights that can be drawn from the results are that:Regarding selected studies on attacks categorized in Device categories and in the Networks category. Buffer overflows, ddos vulnerabilities and backdoors are the most common vulnerabilities found in devices, and network vulnerabilities are directly related to attacks on devices and services.Regarding selected studies on vulnerabilities categorized in Regarding selected studies on defenses categorized in Enable intra-exchange and inter-exchange of industrial data, taking into account the full value of exchanged data;Enable secure data exchange within particular corporation scopes and time frames, while maintaining the control on the data providers;Exercise control of data over devices and the Internet, taking into account industrial spaces and borders;Massive data sharing within sectors or groups of companies;Allow secure data ownership transfer between sectors or groups of companies;Perform real-time analysis of massively generated data;Enforce personal data privacy mechanisms when sharing data between companies;Enable sharing of secure and trusted data based on standards;Share information in an environment of trust between producers and consumers;Allow interoperability for business ecosystems;Enable data sharing between companies to enhance corporation sustainability;Enforce self-determined control of data use (data sovereignty) while offering new ”smart services” and innovative business processes across companies and industries.From the results obtained regarding data sovereignty, digital sovereignty and cyber sovereignty, it can be concluded that these areas are very recent. The terms “digital sovereignty” and “cyber sovereignty” were less recurrent and, on the other hand, the term “data sovereignty” seems to be a trend for cybersecurity in the context of Industry 4.0. These concepts, focused on secure sharing and owning data, are being addressed and the research work in this area seems to be growing (by the progress of the published papers), while recent initiatives such as ids and GAIA-X are also being promoted. Thus, the following set of future challenges for corporations and industries can be depicted:Security incidents may impact industries that are progressing and deploying Industry 4.0 concepts and, since recent technologies such as ai or iot are being included in Industry 4.0, the progress of these implementations requires constant surveillance regarding cybersecurity. At the same time, it is also important to forecast cybersecurity-related challenges and trends to better build and adapt these implementations to possible future attacks and their impacts.This paper reviews recent research efforts on attacks, vulnerabilities, and defenses in Industry 4.0 implementations, and highlights security-related topics and challenges that seem to be surging in this area. The contributions of this paper can be divided in the following: (1) it presents a systematic survey based on a general and abstract analysis, (2) it analyzes in detail and categorizes selected studies on attacks, vulnerabilities and defenses mechanisms, and (3) it provides a discussion on recent challenges and trends regarding these areas on Industry 4.0.From the systematic review results we concluded that attacks, defenses and vulnerabilities in Industry 4.0 implementations are increasing and capturing researcher attention. When categorizing selected studies, we concluded that the greatest number of attacks are network-based attacks, such as dos and mitm attacks, exploiting vulnerabilities in Industry 4.0 networks and devices. Additionally, it is highlighted that topics related to digital sovereignty and mainly data sovereignty appear to be a trend in this area, and ids and GAIA-X are recent initiatives intended to face the challenges related to the data sovereignty topic.


Heart failure (HF) with preserved ejection fraction (HFpEF) is a major public health problem with cases projected to double over the next two decades. There are currently no US Food and Drug Administration–approved therapies for the health-related outcomes of HFpEF. However, considering the high prevalence of this heterogeneous syndrome, a directed therapy for HFpEF is one the greatest unmet needs in cardiovascular medicine. Additionally, there is currently a lack of mechanistic understanding about the pathobiology of HFpEF. The phenotyping of HFpEF patients into pathobiological homogenous groups may not only be the first step in understanding the molecular mechanism but may also enable the development of novel targeted therapies. As obesity is one of the most common comorbidities found in HFpEF patients and is associated with many cardiovascular effects, it is a viable candidate for phenotyping. Large outcome trials and registries reveal that being obese is one of the strongest independent risk factors for developing HFpEF and that this excess risk may not be explained by traditional cardiovascular risk factors. Recently, there has been increased interest in the intertissue communication between adipose tissue and the heart. Evidence suggests that the natriuretic peptide clearance receptor (NPR-C) pathway may play a role in the development and pathobiology of obesity-related HFpEF. Therefore, therapeutic manipulations of the NPR-C pathway may represent a new pharmacological strategy in the context of underlying molecular mechanisms. Heart failure (HF) with preserved ejection fraction (HFpEF) is a major public health epidemic with an economic impact that is at least as great as that of HF with reduced ejection fraction (HFrEF) . HFpEF iThe phenotyping of HFpEF patients into pathobiological homogenous groups has been recently suggested for the development of targeted therapies . There aThere has recently been an increased interest in the intertissue communication between adipose tissue and the heart . AlthougFor this narrative review, pertinent studies were retrieved from seven electronic databases  using common keywords applied in the field of obesity, diabetes, metabolic syndrome, HFpEF, and NPR-C. The author also examined the complete list of relevant references in recent publications in English (both human and studies) on the topics investigated. Given the design of this article as a narrative review, no formal criteria for study selection or quality assessment were applied.i) and may reduce adenylyl cyclase activity and intracellular cAMP levels , including the B-type (BNP), may be mediated through attachment to three distinct NP receptors A, B, and C  . The binP levels .The relationship between the NP system and adipose tissue is well established. Circulating BNP levels appear to be lower in obese patients than in their normal-weight counterparts. This is even more evident in HFpEF, whereby obese HFpEF patients typically have lower circulating BNP levels than their normal-weight counterparts . This coAdipose tissue is the second largest expression site of NPR-C after the kidney, and the NPR-C signaling pathway within adipose tissue is the main target for endogenous agonists . ImpaireThe obesity-HFpEF phenotype is growing increasingly more common, with more than 1.8 million patients in the United States currently diagnosed with this clinical entity . The obeEvidence suggests that NPR-C gene expression in human adipose tissue may be greater in obese hypertensive than in obese normotensive individuals . In obesIn summary, an impaired NPR-C pathway may contribute to the susceptibility of postmenopausal women in developing hypertension and plays an essential role in the pathobiology of HFpEF in women. It would be interesting to investigate, at least in this patient cohort, the effects of long-term treatment with estrogen on the cardiovascular outcomes.–/– mice greatly expedited disease progression. Specifically, the mice lacking NPR-C that were treated with Ang II showed a much more severe decline in cardiac function and decompensation into overt HFrEF as indicated by ventricular dilatation and reductions in EF and FS  was able to largely prevent or slow the development of diastolic dysfunction, and cANF cotreatment did not prevent the increase in systolic blood pressure elicited by Ang II . Similarroblasts , some ofroblasts . In lineroblasts .The development of diastolic dysfunction appeared independent of age, gender, body weight, and systemic hypertension in subjects homozygous for the altered NPR3 genotype with an odds ratio of 1.9, which is similar to that of systemic hypertension, a major modifiable traditional cardiovascular risk factor for diastolic dysfunction . The potTaken together, low NPR-C activity may remove the brake on cardiac hypertrophy, thereby inducing cardiac fibrosis and remodeling and leading to diastolic dysfunction, the major cardiac functional deficit in HFpEF.Coronary artery disease (CAD) is common and extensive in patients with HFpEF, with a prevalence approaching 40–50% . In addiNPR-C that are specifically associated with CAD  injury] .4–23 elicits the potent relaxation of coronary arteries as well as offers a protective mechanism against I/R injury with suppression of both infarct size and myocardial dysfunction. Furthermore, the administration of cANF4–23 during the reperfusion period alone may also protect against I/R injury, suggesting that the activation of NPR-C signaling may prove beneficial in patients presenting with an acute coronary ischemic event  synthase inhibition . In factMaintaining the patency and integrity of the coronary arteries after successful restoration of coronary blood flow is of paramount importance. In patients undergoing PCI, reocclusion of the coronary artery often occurs within 6 months . For theThe beneficial effects of NPR-C agonists may also be applied to patients undergoing coronary artery bypass grafting (CABG). In patients undergoing CABG, the saphenous vein graft (SVG) is still commonly used as graft material; however, the 1-, 5-, and 10-year postoperative SVG patency rates may be 93, 74, and 41%, respectively . FurtherCompared to non-obese HFpEF patients, obese HFpEF patients may display increased epicardial fat thickness . ClinicaAlthough it may be premature to make any final conclusions about cause and effect, some observations support the notion that low NPR-C levels may promote plaque vulnerability/instability and progression in patients with atherosclerotic cardiovascular disease . One appAtrial fibrillation (AF) is highly prevalent and commonly occurs in the setting of HFpEF and hypertension , conditi–/– mice (and NPR-C+/+ littermates) with saline or Ang II   . Furthertermates . Intracatermates . Strikintermates .–/– mice may be caused primarily by enhancing fibrosis in the atria, suggesting that NPs may act upon NPR-C in cardiac fibroblasts to regulate extracellular matrix deposition  levels, and NPR-C expression, suggesting that hyperinsulinism and altered glucose metabolism may contribute, at least in part, to the elevated NPR-C expression in individuals with high BMI. There is a gradual and progressive increase of NPR-C transcripts in adipose tissue when an individual is transitioning from normal glucose tolerance to type 2 diabetes mellitus (T2DM)  . Althougs (T2DM) .NPRC mRNA and improves insulin sensitivity in patients with T2DM or metabolic syndrome. Skeletal muscle NPR-C has also been shown to be positively related to fasting blood glucose, insulin, and HbA1c, further suggesting that a functional NPR-C pathway is required for insulin sensitivity and blood glucose control (In addition, hyperinsulinemic states, as found in patients with impaired glucose tolerance/insulin resistance due to metabolic syndrome or diabetes, may up-regulate subcutaneous NPR-C gene expression in a glucose-dependent manner . Kovacov control . In a stAlthough it may be premature to make any final conclusions about cause and effect, the above findings suggest that the NPR-C pathway may be dysregulated in the context of metabolic disorders such as obesity, insulin resistance, and T2DM . The obsEven though obesity is a strong risk factor for HF in the general population, the “obesity paradox” refers to the fact that obese patients with established HF tend to have better long-term prognosis than non-obese patients . PatientAlthough the association between obesity and HFpEF is well known, the pathophysiology of weight-related changes on the outcome of patients with HFpEF is still a matter of debate. Unfortunately, most obesity paradox studies used BMI cut points to classify subjects as normal, overweight, or obese . HoweverAlthough the potential underlying mechanisms of the obesity paradox have been extensively studied , little Some evidence suggests that the prognostic impact of increased body fat mass may also be lost systematically as the disease progresses, and HF severity may overcome percent body fat in the prediction of short-term cardiovascular events . Indeed,The activation of the sympathetic nervous system is one of the compensatory mechanisms mounted by the body to maintain cardiac output in individuals with HF. Evidence suggests that an augmented activation of the sympathetic system may reduce the levels of NPR-C in a time- and dose-dependent manner by decreasing the transcriptional rate of the NPR-C gene . The catThe above findings suggest that enhancing the NPR-C pathway may represent an attractive therapeutic strategy to reduce body wasting, increase the ability to tolerate higher HF therapeutic doses, and improve HF outcomes.Natriuretic peptide clearance receptor signaling may be central to the pathobiology of HFpEF in general and particularly to the obesity-HFpEF phenotype (as illustrated in The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are highly immunogenic. The most recent HPV vaccine, Gardasil-9, protects against HPV types associated with ~90% of cervical cancer (worldwide). Thus, ~10% of HPV-associated cancers are not protected by Gardasil-9. Although this is not a large percentage overall, the HPV types associated with 10% of cervical cancer not protected by the current vaccine are significantly important, especially in HIV/AIDS patients who are infected with multiple HPV types. To broaden the spectrum of protection against HPV infections, we developed mixed MS2-L2 VLPs (MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs) in a previous study. Immunization with the VLPs neutralized/protected mice against infection with eleven high-risk HPV types associated with ~95% of cervical cancer and against one low-risk HPV type associated with ~36% of genital warts & up to 32% of recurrent respiratory papillomatosis. Here, we report that the mixed MS2-L2 VLPs can protect mice from three additional HPV types: HPV51, which is associated with ~0.8% of cervical cancer; HPV6, which is associated with up to 60% of genital warts; HPV5, which is associated with skin cancers in patients with epidermodysplasia verruciformis (EV). Overall, mixed MS2-L2 VLPs can protect against twelve HPV types associated with ~95.8% of cervical cancers and against two HPV types associated with ~90% of genital warts and >90% recurrent respiratory papillomatosis. Additionally, the VLPs protect against one of two HPV types associated with ~90% of HPV-associated skin cancers in patients with EV. More importantly, we observed that mixed MS2-L2 VLPs elicit protective antibodies that last over 9 months. Furthermore, a spray-freeze-dried formulation of the VLPs is stable, immunogenic, and protective at room temperature and 37 °C. Human papillomaviruses (HPVs) are non-enveloped, double-stranded DNA viruses ,2. ThereCurrently, three prophylactic vaccines  have been approved to protect against HPV infections. The vaccines are based on virus-like particles (VLPs) derived from the expression of the major capsid protein, L1 . L1-baseThe HPV capsid is made up of two capsid proteins: the major capsid protein (L1) and the minor capsid protein (L2). The capsid proteins assemble to form a virus particle or VLP as follows: first, the L1 protein forms pentamers, and the L2 protein is inserted  at the vertex of each pentamer. Seventy-two copies of the pentamers then assemble to form a virus particle or VLP . During Escherichia coli bacteria were separately transformed by heat shock with the two recombinant plasmids (expressing MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs). Cultures of transformed bacteria were grown at 37 °C until an optical density (OD)600 of 0.6, and the cultures were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 3 h. The bacteria were pelleted and lysed with 0.2% lysozyme solution (for MS2-L2 consL2 (69-86)) or 10 mM borax solution (for MS2-31L2/16L2 VLPs). The VLPs were precipitated using 50% (w/v) ammonium sulfate and purified by gel filtration using Sepharose CL-4B columns.Plasmids encoding recombinant MS2-L2 proteins have previously been described  15]. Th. Th15]. w/v MTDL excipient solution, which contained mannitol , trehalose , dextran , and L-leucine . The VLPs were added at a concentration of 8% w/w to the MTDL excipients, and the VLPs-excipients were then spray-freeze-dried (SFD) [Spray-freeze-dried MS2 VLPs or mixed MS2-L2 VLPs powders (filled in capsules without adjuvants) have previously been described . Brieflyed (SFD) . SFD VLP450nm using the Synergy LX multimode reader . Antibody titers were determined by considering the reciprocal of the highest sera dilutions at which the reactivity of experimental sera was greater than 2-fold compared to control sera at the same dilution.Animal work was conducted following Michigan Technological University Institutional Animal Care and Use Committee guidelines (Protocol Number: L0264). Groups of 5 female BALB/c mice were immunized intramuscularly with 10 μg of mixed MS2-L2 VLPs (5 μg each of MS2-31L2/16L2 VLPs and MS2-consL2 (69-86)) in the presence of alum hydroxide . 10 μg amounts of reconstituted SFD-mixed MS2-L2 VLPs stored at room temperature or 37 °C were also used to immunize mice in the presence of alum hydroxide. Control mice were immunized with 10 μg each of Gardasil-9, MS2 VLPs, and spray-freeze-dried MS2 VLPs . All immunizations were performed twice at two-week intervals. To assess the longevity of immune responses, 6 female BALB/c mice were immunized, thrice, with 10 μg of mixed MS2-L2 VLPs with alum at two-week intervals. The mice were immunized thrice because the concentration of each of the L2 VLP in the mixture of VLPs was only 5 μg. Whole blood was collected 2 weeks after the last immunizations (to assess antibody titers in sera) except from the groups of mice that were immunized with VLPs for longevity studies. In these groups of mice, sera were collected monthly. IgG antibody titers in sera were conducted by peptide enzyme-linked immunosorbent assay (ELISA) using the following peptides as target antigens: (i) 16L2 peptide, (ii) 31L2 peptide, and (iii) HPV consL2 (69-86) . Brieflyw/v). The gradient was spun for 17 h (4 °C) at 20,000 rpm, and a band corresponding to HPV PsVs was collected. HPV PsVs contain pClucf plasmids, which can be used to assess infectivity based on the expression of the reporter genes (luciferase and green fluorescence protein) in infected cells. Infection studies were conducted as previously described [6 infectious units (IUs) of PsVs as described previously. Forty-eight hours after PsV infection, mice were genitally instilled with 0.4 mg of luciferin (a substrate for luciferin). Mice were imaged using IVIS Spectrum at one-minute exposure to capture the intensity of luciferase expression . Average radiance (p/cm2/sr) was determined by drawing equally sized regions of interest surrounding the site of PsV administration.HPV pseudoviruses (PsVs) representing HPVs 5, 6, and 51 were made and purified by cesium chloride gradient ultracentrifugation as described previously ,23. In bescribed ,24,25. Ct-test and an unpaired one-tailed t-test, respectively.Statistical analysis of ELISAs and HPV PsV challenge studies were performed using an unpaired two-tailed 3) were similar to those from our previous study [We previously showed that non-SFD-mixed MS2-L2 VLPs are immunogenic . Howeverus study . High-ti3) could offer protection against HPV pseudovirus infection, immunized mice were genitally infected with HPV PsV6. As shown in To assess at what temperature and for how long the SFD-mixed MS2-L2 VLPs can be stored without refrigeration, the SFD VLPs were stored at room temperature for 6 months and at 37 °C for 2 months, and their integrities were assessed using TEM. As shown in To assess how long the immune responses elicited by mixed MS2-L2 VLPs would last, mice were immunized with non-SFD-mixed MS2-L2 VLPs, and antibody titers were monitored monthly for 10 months. As shown in Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are very immunogenic. However, they protect mostly against HPV types included in the vaccines. For example, Gardasil-9, which has the most HPV VLP types  included in the vaccines, protects against HPVs associated with ~90% of cervical cancer, 80–85% of HPV-associated vaginal cancer, 85–90% of HPV-associated vulvar cancer, 90–95% of HPV-associated anal cancer, ~90% of genital warts, and ~86% of HPV-associated penile cancer ,27,28,29p = 0.1434)  . For exa 0.1434) . Three o 0.1434) C. We areIn summary, mixed MS2-L2 VLPs are an ideal candidate HPV vaccine that should be explored further. They are highly immunogenic. Non-SFD-mixed MS2-L2 VLPs can protect against HPV PsVs5, 6, and 51. This is in addition to protection against HPV PsV11, 16, 18, 31, 33, 35, 39, 45, 53, 56, 52, and 58) in our previous studies ,20. Thus