BTBD9 and MEIS1 genes, have been linked to an increased risk of RLS initiation and augmentation. Additionally, circadian rhythm, which controls the sleep–wake cycle, may also contribute to the worsening of RLS symptoms and the development of augmentation. Recently, Vitamin D deficiency has been suggested to be involved in RLS augmentation. Based on these findings, we propose that the progressive reduction of selective receptors, influenced by various pathological factors, reverses the overcompensation of the dopamine intensity promoted by short-term, low-dose dopaminergic therapy in the development of augmentation. More research is needed to uncover a deeper understanding of the mechanisms underlying the RLS symptom and to develop effective RLS augmentation treatments.Long-term drug treatment for Restless Legs Syndrome (RLS) patients can frequently result in augmentation, which is the deterioration of symptoms with an increased drug dose. The cause of augmentation, especially derived from dopamine therapy, remains elusive. Here, we review recent research and clinical progress on the possible mechanism underlying RLS augmentation. Dysfunction of the dopamine system highly possibly plays a role in the development of RLS augmentation, as dopamine agonists improve desensitization of dopamine receptors, disturb receptor interactions within or outside the dopamine receptor family, and interfere with the natural regulation of dopamine synthesis and release in the neural system. Iron deficiency is also indicated to contribute to RLS augmentation, as low iron levels can affect the function of the dopamine system. Furthermore, genetic risk factors, such as variations in the  The disorder affects an estimated 1–15.3% of the population, with about 1–3% of people overall experiencing severe and frequent symptoms . DespiteDespite the advancements in the development of drug targeting various transmitter pathways, dopaminergic agents(levodopa and dopamine agonists) have remained the mainstay of treatment for restless legs syndrome for the past four decades –10. The “Augmentation” refers to an iatrogenic exacerbation of RLS symptoms, as characterized by earlier occurrence of symptoms in the afternoon than before treatment initiation, the spread of symptoms to the upper limbs, and a reduced latency until symptoms manifest during periods of rest. Another recognizable characteristic of augmentation is the paradoxical worsening of symptoms upon increasing the dose of dopamine agonists. The clinical phenomenon of augmentation was first observed during long-term levodopa treatment and was suggested to be a result of the treatment itself . Augment2.2.1.Several studies have supported the increased dopamine synthesis and secretion on the presynaptic surface in RLS patients –32. Howe2.2.The interaction between dopamine receptors within the dopamine receptor family and other neurotransmitter systems is implicated in the development of RLS augmentation. Specifically, high dopamine concentrations, possibly resembling the hyperdopaminergic state observed in patients with augmentation, target excitatory dopamine 1 receptors (D1Rs), which sustaining locomotor-like activity in the isolated spinal cord, potentially contributing to augmentation . Activat2.3.Desensitization of dopamine receptors may also contribute to the development of augmentation. Like other G protein-coupled receptors (GPCRs), dopamine receptors regulate signal transduction by receptor desensitization . Recent 3.The role of iron in RLS pathogenesis has been established through numerous studies, as well as the positive effect of iron supplementation on RLS symptoms. However, the relationship between iron metabolism and RLS augmentation is still unclear. The severity of RLS symptoms at baseline and with augmentation has been associated with low ferritin levels, which suggests a deficiency in mobilizable iron stores , 49. Bes4.MEIS1 and BTBD9 loci have a significant genetic association with RLS (BTBD9 gene was significantly associated with serum ferritin levels in female blood donors (MEIS1 gene and an increase in the expression of H-ferritin, L-ferritin, and divalent metal transporter-1 RNA has been uncovered in the thalamus (The contribution of genetic background to RLS augmentation is poorly understood. The significant physiological and pathological association between genetic risk factors, dopamine dysfunction, and iron metabolism suggests that RLS risk factors contribute to the development of augmentation. The with RLS  and are with RLS . Overexpwith RLS . A previd donors . On the thalamus .BTBD9 and its homolog are involved in the transcriptional and cellular regulation of dopamine D2Rs and D3Rs, which are the main targets of dopamine agonists. The internalization of D2R is modulated by DNM-1 (BTBD9 systematic knockout mice. The expression of DNM-1 was found to be enhanced in the BTBD9 complete knockout mice compared to wild-type mice that were euthanized at midnight (BTBD9 complete knockout mice can be alleviated by D2/D3 agonists (BTBD9 mutant flies showed a significant decrease in brain dopamine levels and an incoordinate sleep phenotype, which was majorly rescued by giving pramipexole (BTBD9 deficiency, the activity of the D1R-mediated dopamine system may have reached its peak, which indicated a weakened D2R signaling but an increased D1R signaling in RLS syndrome (BTBD9 loci may have a tendency toward augmentation in the pathological process.by DNM-1 , which smidnight . The symagonists . A reseamipexole . In contmipexole . In bothmipexole , 65. Undsyndrome , similarsyndrome . Therefo5.Vitamin D deficiency has been shown to correlate with RLS symptoms on serum vitamin D level , 68. Vit6.RLS symptoms are more apparent at night, with a higher occurrence reported in the evening and night hours . Indeed,D1R, D2L, and D2S mRNA levels were all decreased during the locomotion time compared with the sleep time (D2R mRNA was increased in the striatum of BTBD9 complete knockout at midnight, indicating that the effect of BTBD9 deficiency on the D2R mRNA level is regulated by circadian rhythm (BTBD9 mutant mice exhibit abnormal sleep architecture, which is reminiscent of RLS patients (BTBD9 variation impacts circadian. On the other hand, MEIS1 haploinsufficiency was associated with a sex-dependent increase in activity during the onset of rest, similar to the circadian rhythm of RLS symptoms observed in human patients (Pathological factors involved in augmentation, such as abnormal dopamine system dynamics, genetic risk factors expression, and iron concentration, display diurnal rhythmicity. This view suggests that the timing of dopamine agonist treatment may influence development of RLS augmentation through pathological networks. Dopamine release has an unambiguous circadian activity pattern of decreasing in the evening and night and increasing in the morning –83. In weep time . The levn rhythm . Arrhythn rhythm . Signifin rhythm . Diurnaln rhythm . Researcn rhythm . In termpatients , it is ypatients .7.Overall, the effect of dopamine agents, potentially accompanied with iron deficiency, genetic risk factors, vitamin D deficiency and effect of circadian rhythms, contributes to the attenuation of dopamine signaling in patients with augmented RLS. RLS patients may exhibit impaired postsynaptic response to increased dopamine stimulation after long-term administration of low-dose dopamine agents, possibly due to dopamine receptor desensitization and interaction of dopamine receptors. When dopamine signaling is reduced during dusk and night, this insufficient postsynaptic dopamine signaling causes augmented RLS symptoms. More specifically, despite an overall increase in dopamine secretion in RLS patients, continuous treatment with dopamine agents may desensitize selective dopamine receptors, with a gradual decrease in peak dopamine efficiency over time. This leads to a deficit in dopamine signaling in the evening and at night compared with healthy people. As a result, the circadian attenuation of dopamine signaling in the evening and at night in augmented RLS patients disrupts normal sleep homeostasis, with the symptom of fragmented sleep at rest in these patients. The dopamine drug-induced incremental activation of dopamine signaling exacerbates the downregulation of overall post-synaptic dopamine receptors, thus worsening the RLS disease and expanding the symptoms .8.Augmentation is one of the greatest threats to RLS patients in the context of the widespread usage of dopamine agents. The pathological mechanism behind this symptom is unclear and needs to be elucidated. Current clinical diagnoses and research findings highlight the potential mechanisms linking dopamine dysfunction, circadian regulation, iron deficiency, vitamin D deficiency and genetic risk factors to the development of augmentation. However, a multi-perspective investigation in the field is lacking. In addition, the role of other transmitter signaling pathways in the development of augmentation remains vague. Further careful and long-term research is necessary to fully understand the underlying mechanisms of augmentation in RLS patients.PZ contributed to the original concept of the article, did the literature search, wrote the original version of the manuscript, drew the figure and reviewed it as it progressed. TW, LZ, and FG contributed to the original concept of the manuscript and reviewed the manuscript. All authors contributed to the article and approved the submitted version.


The ethylene propylene diene monomer (EPDM) is utilized in high voltage direct current (HVDC) cable accessories due to its exceptional insulation properties. The microscopic reactions and space charge characteristics of EPDM under electric fields are studied using density functional theory. The results indicate that as the electric field intensity increases, the total energy decreases while the dipole moment and polarizability increase, leading to a decrease in the stability of EPDM. The molecular chain elongates under the stretching effect of the electric field and the stability of the geometric structure decreases, resulting in a decline in its mechanical and electrical properties. With increased electric field intensity, the energy gap of the front orbital decreases, and its conductivity improves. Additionally, the active site of the molecular chain reaction shifts, leading to different degrees of hole trap and electron trap energy level distribution in the area where the front track of the molecular chain is located, making EPDM more susceptible to trapping free electrons or injecting charge. When the electric field intensity reaches 0.0255 a.u., the EPDM molecular structure is destroyed, and its infrared spectrum undergoes significant changes. These findings provide a basis for future modification technology, and theoretical support for high voltage experiments. A high voltage direct current (HVDC) cable transmission system possesses several advantages, including low cost, reactive power compensation, and compatibility , so as tIn the pursuit of advancements in high voltage grade transmission lines, the design and implementation of high voltage cable accessories constitutes a crucial aspect . StudiesMolecular simulation technology, as an emerging research tool, offers a unique capability to simulate and to calculate experiments that are impossible to achieve under real experimental conditions . FurtherIn order to obtain the weak sites inside the material, and to provide theoretical support for high-voltage experimental evidence, and avoid blindly adding electric fields in high-voltage experiments, this study uses quantum chemistry methods for theoretical research. The method studied in this paper is based on density functional theory. The process begins with the establishment of an EPDM molecular model and its geometric optimization, which is carried out using the Gaussian 09W software. Subsequently, the electrostatic potential surface of the EPDM molecule is analyzed through the use of Multiwfn  softwareIn this experiment, the ethylene propylene diene monomer (EPDM) material utilized is composed of ethylene, propylene, and a third monomer, namely ethylene-norbornene (ENB), with an unsaturated olefin bond on its side chain that accelerates the vulcanization rate. The vulcanized EPDM boasts high strength, filling, and extrusion performance , and is H of ENB-EPDM molecules under an external electric field can be expressed as Equation (1). H0 is the Hamiltonian of the molecule without an electric field; Hint is the Hamiltonian when the external electric field interacts with the molecular system. In the case of dipole approximation, the interaction energy between the molecular system and the external electric field can be expressed as Equation (2). μ is the electric dipole moment and F is the external electric field force.The Hamiltonian 11 V/m) of the molecular chain, respectively, and the optimization and single point energy calculation are conducted to study the microscopic mechanism of the electrical aging of the ENB-EPDM materials. All simulation calculations are carried out in the Gaussian 09W software package. Multiwfn 3.7 software  between the leftmost C1 atom and the rightmost C30 atom of the molecular chain under different external electric fields (0~0.0255 a.u.) is shown in After optimizing the ENB-EPDM molecule to obtain a stable spatial structure, the variation trend of distance Under the long-term polarization of electric field, the geometry of the ENB-EPDM molecular chain will be gradually stretched. When the internal field strength reaches a certain value, the molecular chemical bond will break, resulting in the breakdown of insulation. The space charge distortion in the molecular chain under the continuous action of the electric field is the main reason for the increase in the internal field intensity in the insulating material.According to the frontier orbital theory, the energy of the highest electron occupied orbital (HOMO) in a molecule is the highest, the electron is the least bound, and the transition is the most likely to occur. The lowest unoccupied orbital (LUMO) has the lowest energy of all unoccupied orbitals and accepts electrons the most easily. The energy gap (Eg) between the HOMO and the LUMO of the front orbital is a measure of the electron’s ability to move from an occupied orbital to an empty one, and it is often used to measure the molecule conductivity. The smaller Eg is, the better the conductivity of the molecule is, and the easier it is for electrons to transition from the HOMO to LUMO orbitals. As shown in In order to analyze the microscopic variation characteristics of the space charge inside the ENB-EPDM under the continuous electric field, the changes of trap energy level and the orbital cloud map can be used to discuss. It can be seen from the distribution of the molecular orbits (MOs) map that before the electric field is applied, the HOMO orbital is mainly concentrated to the left end of the molecular chain, which is easily attacked by electrophile reagents and electrophilic reactions that occur. Additionally, the LUMO orbital is mainly concentrated to the left end of the molecular chain, which is vulnerable to nucleophile attack and nucleophilic reaction. However, the effect of the external electric field makes the distribution of the molecular front track change obviously, and the reaction active site of molecule also changes accordingly, as shown in As can be seen from the energy level distribution diagram in The results calculated using the Gaussian software are then processed using Multiwfn software using the default parameters, with each orbital having a strength value of 1, and the degeneracy using the default 0.005 eV threshold. −1 is the strongest at the end of the molecular chain. With the increase in the external electric field, the corresponding absorption peak appears to split. This indicates that the nuclear skeletons and electronic structures of the ENB-EPDM molecules have changed greatly under the action of a strong electric field, which leads to the enhancement of the infrared activity of vibration modes in more intervals, and the shift of resonant frequency. When the electric field intensity continues to increase, the virtual frequency appears in the infrared spectrum of the ENB-EPDM molecule, indicating that the molecular structure is not stable. In this unstable state, the structure of the ENB-EPDM molecule is easily destroyed. The inflection point where the internal molecular structure of the ENB-EPDM dielectric changes is the starting point of the electrical branch aging of the insulating materials. The internal molecular structure of ENB-EPDM is destroyed, and a large number of free radicals are formed. Free radicals will attack groups on other chains, resulting in bond breaking and the cracking of more molecular chains. The breakage of the molecular chain must lead to the destruction of the ENB-EPDM material, thus reducing the electrical strength of the cable, and finally the polymer insulation breakdown phenomenon. It is of great significance for studying the mechanism of electrical aging.Finally, Multiwfn software is used to further process the results calculated using Gaussian software. The default parameter is used, and the frequency correction factor is 0.9614. The infrared spectra obtained via a simulation of the ENB-EPDM molecules at different electric field intensities are shown in In this study, the molecular model of ENB-EPDM was constructed using a molecular simulation approach based on quantum mechanics to analyze and to calculate the influence of external electric fields on the internal microscopic characteristics of ENB-EPDM molecules. The results reveal changes in the insulation properties of ENB-EPDM materials at the molecular level. The key findings are:(1) The electrostatic potential surface analysis of ENB-EPDM molecules shows that the C=C bond and the H atoms near the carbon ring have strong reactivity and are prone to reaction, providing evidence for the initial bond breaking of ENB-EPDM under strong electric field conditions, and a theoretical basis for modifying ENB-EPDM insulation materials.(2) With an increasing external electric field, the dipoles of the ENB-EPDM molecules will rotate in a directional manner, forming equivalent polarized space charges inside the ENB-EPDM molecules. The stronger the electric field, the higher the polarization rate and space charge density, leading to an increase in molecular activity and instability, or even the destruction of the molecular spatial structure. The analysis of the space charge characteristics shows that the energy gap decreases, leading to better molecular conductivity. When the energy gap decreases to a certain level, the chemical bond breaks. The electric field changes the reactive sites of the molecular chain, leading to electrophilic and nucleophilic activities at both ends of the chain, and the uneven distribution of trap energy levels along the front track of the molecular chain, resulting in a distorted local field strength inside the molecule.(3) The inflection point in the molecular structural characteristics is the starting point of electric branch formation. When the electric field intensity reaches 0.0255 a.u., ENB-EPDM molecules undergo electrical breakdown, with the hydrogen atom at the end of the molecular chain breaking first and generating free radicals, which in turn affects the internal microscopic characteristics of ENB-EPDM. This results in changes in the corresponding infrared spectrogram, providing a theoretical basis for evaluating the state of insulation. This is due to the increasing voltage level of the transmission network, the high requirements for insulation materials, and the breakdown of the ultra-high voltage of power failures. Based on the above situation, molecular simulation can provide theoretical support for the subsequent engineering practical tests of this project. It is targeted and avoids blindly carrying out experiments. Through the theoretical research of molecular simulation, it will be able to save the considerable costs of a strong electric field. It can also provide support for our subsequent material modification technology.


The generation of induced pluripotent stem cells has opened the field of study for stem cell research, disease modeling and drug development. However, the epigenetic signatures present in somatic cells make cell reprogramming still an inefficient process. This epigenetic memory constitutes an obstacle in cellular reprogramming. Here, we report the effect of hydralazine (HYD) and valproic acid (VPA), two small molecules with proven epigenetic activity, on the expression of pluripotency genes in adult (aHF) and neonatal (nbHF) human fibroblasts.aHF and nbHF were treated with HYD and/or VPA, and viability and gene expression assays for OCT4, NANOG, c-MYC, KLF4, DNMT1, TET3, ARID1A and ARID2 by quantitative PCR were performed. aHF and nbHF were transfected with episomal plasmid bearing Yamanaka factors  and exposed to HYD and VPA to determine the reprogramming efficiency. Methylation sensitive restriction enzyme (MSRE) qPCR assays were performed on OCT4 and NANOG promoter regions. Immunofluorescence assays were carried out for pluripotency genes on iPSC derived from aHF and nbHF.HYD upregulated the expression of OCT4 (2.5-fold) and NANOG (fourfold) genes but not c-Myc or KLF4 in aHF and had no significant effect on the expression of all these genes in nbHF. VPA upregulated the expression of NANOG (twofold) in aHF and c-MYC in nbHF, while it downregulated the expression of NANOG in nbHF. The combination of HYD and VPA canceled the OCT4 and NANOG overexpression induced by HYD in aHF, while it reinforced the effects of VPA on c-Myc expression in nbHF. The HYD-induced overexpression of OCT4 and NANOG in aHDF was not dependent on demethylation of gene promoters, and no changes in the reprogramming efficiency were observed in both cell populations despite the downregulation of epigenetic genes DNMT1, ARID1A, and ARID2 in nbHF.Our data provide evidence that HYD regulates the expression of OCT4 and NANOG pluripotency genes as well as ARID1A and ARID2 genes, two members of the SWI/SNF chromatin remodeling complex family, in normal human dermal fibroblasts.The online version contains supplementary material available at 10.1186/s13287-023-03268-w. Induced pluripotent stem cells (iPSC) are derived from somatic cells that have been reprogrammed to an embryonic-like stage . Somaticγ-aminobutyric acid (GABA) in the brain or by altering the properties of sodium channels [Drugs with regulatory effects on the epigenome, termed “epigenetic drugs,” have been identified. Epigenetic drugs are mainly divided into two categories: the ones that modify DNA methylation patterns and those that inhibit histone deacetylases . Within channels , 12. Nevchannels , 14. TheHYD hydrochloride  and VPA sodium salt  were purchased from Sigma-Aldrich . For assays, HYD and VPA were diluted in culture medium from an aqueous stock solution.50 values were determined with the dose–response curve of each drug at 72 h according to the GraphPad software method (log(inhibitor) vs. normalized response).Human adult dermal fibroblasts (aHF) (ATCC PCS-201–012) and neonatal foreskin BJ fibroblasts (nbHF) (ATCC CRL-2522), were cultured and cell viability was assessed according to conditions previously reported . The IC5−ΔΔCT [For RNA extraction and reverse transcription, we followed the methods of Aguirre-Vázquez et al. . The fun−ΔΔCT  method. MspI/HpaII)  according to the manufacturer’s instructions was performed. DNA digestion with MspI and HpaII enzymes or the undigested control reaction was carried out in 20 μL with 200 ng of gDNA for 4 h at 37 °C. Then, the samples were incubated at 90 °C for 10 min. Subsequently, qPCR was performed by technical duplicates from three biological replicates using 1 μL of digested (MspI/HpaII) or undigested genomic DNA in 10 μL volume using the EXPRESS SYBR GreenER qPCR SuperMix Universal (Invitrogen) and 0.35 nM of primer forward/reverse. The primer sequences used in this assay are shown in Table HpaII Rx–Ct undigested Rx) × 100. Validation experiments were performed according to the manufacturer’s instructions.Genomic DNA from aHF and nbHF were extracted with Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s recommendations. Afterward, the analysis of DNA methylation with the EpiJET DNA Methylation Analysis Kit (6 cells (between passages 6 and 10) were transfected with a 2.5 µg mix of each vector using a Neon Transfection System (Invitrogen). The conditions for aHF were 1800 V, 20 ms with one pulse, and for nbHF, they were 1650 V, 10 ms with three pulses. After transfection, cells were cultured for 7 days in reprogramming medium in the presence or absence of 30 µM HYD. The reprogramming medium was formulated with DMEM high glucose supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum (Gibco), 10% KnockOut Serum Replacement (Gibco), 1 mM sodium pyruvate (Corning), 1% non-essential amino acids, 3 µM CHIR99021(Sigma-Aldrich) and 0.5 µM A83-01 (Sigma-Aldrich). At day 8, cells were recovered and seeded on mitotically inactivated mouse embryonic fibroblasts (iMEF). Medium was replaced to KnockOut DMEM supplemented with 20% KnockOut Serum Replacement, 2.5 mM Glutagro (Corning), 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol, and 10 ng/mL of basic Fibroblast growth factor (bFGF) (Corning). Colonies were visualized and counted at 25–30 days, and those with characteristics of human ESC-like colonies [Reprogramming of aHF and nbHF was carried out with episomal reprogramming vectors pCXLE-hOCT3/4-shp53, pCLXE-hSK, and pCLXE-hUL . Brieflycolonies , 20 wereiPSC colonies, aHF and nbHF, were cultured in 24-well plates with glass coverslips precoated with 0.5% gelatin according to the previously described culture conditions . For theiPSC derived from aHF and nbHF were characterized by karyotyping through Laboratorios de Analisis Geneticos Especializados Mexico (LAGEM). G-banding in human metaphase chromosomes was analyzed in the ZEISS Axio Imager microscope  using the Ikaros Karyotyping Software ver 5.9.0  with an EC Epiplan Neofluar 100 × objective with correction to infinite coupled to the CoolCube 1—digital high -resolution progressive scan CCD camera (Metasystems), with 1360 × 1024 pixels of resolution.U test using the SPSS v2.0 software and GraphPad Prism 6 . The criterion for significance was set at P < 0.05 in all cases.Data are shown as the means values ± standard error of the mean. All data were analyzed by Mann–Whitney P < 0.05) and 15% (P < 0.05) at 72 and 96 h of HYD-VPA treatment, respectively  from 24 h to just reaching 36% (P < 0.05) at 96 h  increase compared to the untreated group, although these changes were only seen in aHF  and twofold (P < 0.05) in the transcription levels by the individual effect of HYD and VPA in aHF, respectively  and HYD-VPA (P < 0.05) treatments in aHF  and HYD-VPA treatments  assay. Evaluation of 5-mC at three and four specific 5′-CCGG-3′ sites were performed in the proximal promoter region of OCT4 and NANOG, respectively , but this was only observed in nbHF    inhibitor and that downregulation of DNMT1 activity improves the reprogramming efficiency; however, no changes in the number of colonies were observed. Therefore, to ascertain the effect of HYD as an epigenetic drug, we evaluated, by RT-qPCR assays, its effect on the expression of genes related to DNA methylation and genes involved in chromatin structure. First, we confirmed that HYD downregulated DNMT1 expression  Fig. C and 21%05) Fig. D in nbHFWe have found that hydralazine modifies the expression of pluripotency genes in aHF and epigenetic genes in nbHF Fig. , but it The transcriptional regulation of OCT4 and NANOG factors are related to the maintenance of the pluripotency network , oncogenMspI and HpaII enzymes. It is advisable to complement our analyses with techniques that allow to evaluate the total promoter region, such as bisulfite sequencing [Most reports assessing the effect of HYD on transcriptional regulation mainly focus on its repurposing or repositioning activity as adjuvant therapy in cancer treatments. Reactivation of tumor suppressor genes in hypermethylated promoter regions in cancer-derived cell lines has been related to HYD treatment , 25–27. quencing . In addiOur results indicate that the HYD-VPA combination annuls the individual effect of HYD on OCT4 and NANOG gene expression and downregulates the expression of the c-Myc gene in aHF. In this regard, HYD induced a lupus-like phenotype through the inhibition of the ERK pathway, causing downregulation of DNMT1 , 32. IntAlthough HYD did not increase the reprogramming efficiency in our experimental scheme, the use of small molecules capable of inhibiting DNMT1 activity has proven to be an effective strategy to enhance the reprogramming efficiency. Rodriguez-Madoz and colleagues showed that the reversible dual G9a/DNMT1 inhibitor molecule, CM272, enhances the mesenchymal to epithelial transition during the early phase of cell reprogramming . AdditioOther epigenetic regulation mechanisms, independent of DNMT inhibition, have been described for HYD. Dehghan and colleagues reported the correlation between the activation of the histone deacetylase SIRT1 by hydralazine and stress resistance in C. elegans . LikewisAlthough cell culture, treatments, and reprogramming assays were carried out under the same conditions, we observed a difference in the response to the evaluated drugs in both cell populations. We observed these differences in drug sensitivity assays, gene expression analyses, and the total number of iPSC colonies generated between aHF and nbHF. Interestingly, the activity of drug-metabolizing enzymes changes significantly from fetal to adult age , 42. TheFinally, we are aware of the limitations of our experimental strategy when evaluating pluripotency genes in human somatic cell lines. It is necessary to complement our expression analyses with the methodologies proposed by Li et al.  and Hou In this study, we demonstrate that HYD modifies the expression of groups of genes involved in the induction of pluripotency and chromatin remodeling in aHF and nbHF. HYD and VPA have limited effects on the transcriptional regulation of pluripotency genes, which have basal expression levels in our cell models. For this reason, we believe that the effect of both drugs should be evaluated in multipotent, pluripotent stem cells or fibroblasts from other donors to explore whether the effects observed in the gene expression, reprogramming, and epigenetic assays described here occur in other cell models. The main perspective of this work is that treatment with HYD, alone or in combination with other epigenetic modulators, is a promising option to induce the expression of pluripotency genes and chromatin remodeling complexes. Further studies are needed to explore the effect of hydralazine on epigenetic signatures such as acetylation, histone methylation, and the global evaluation of methylated regions in DNA.Additional file1. Figure S1. Chromosomal analysis of iPSC derived from aHF and nbHF.Karyotyping was carried out using standard G-banding in metaphase chromosomes.Analysis revealed normal karyotypes in aHF  and nbHF  at passage 5 and 6, respectively.


To achieve potential financial savings and avoid exposing the patients to unnecessary risk, an optimal diagnostic strategy to identify low risk individual who may derive minimal benefit from further cardiac imaging testing (CIT) is important for patients with stable chest pain (SCP) suggestive of chronic coronary syndrome (CCS). Although several diagnostic strategies have been recommended by the most recent guidelines, few randomized controlled trials (RCTs) have prospectively investigated the actual effect of applying these strategies in clinical practice.OPtimal Evaluation of stable chest pain to Reduce unnecessAry utilization of cardiac imaging TEsting) trial is an investigator-initiated, multicenter, coronary computed tomography angiography (CCTA)-based, 2-arm parallel-group, double-blind, pragmatic and confirmative RCT planning to include 800 subjects with SCP suggestive of CCS. After enrollment, all subjects will be randomized to two arms  on a 1:1 basis. According to each strategy, CCTA should be referred and deferred for a subject in high and low risk group, respectively. The primary (effectiveness) endpoint is CCTA without obstructive coronary artery disease. Safety of each strategy will be mainly assessed by 1-year major adverse cardiovascular event rates.OPERATE (The OPERATE trial will provide comparative effectiveness and safety evidences for two different diagnostic strategies for patients with SCP suggestive of CCS, with the intension of improving the diagnostic yield of CCTA at no expense of safety.ClinicalTrial.org Identifier NCT05640752.The online version contains supplementary material available at 10.1186/s12872-023-03424-3. In daily clinical routine, the evaluation of new-onset stable chest pain (SCP) suggestive of chronic coronary syndrome (CCS) remains a challenge for physicians, in consideration of the decreasing prevalence of coronary artery disease (CAD) and dramatically rising costs related to these patients , 2. AlthDifferent diagnostic strategies have been developed to defer unnecessary CIT. The 2016 U.K. National Institute of Health and Care Excellence (NICE) guideline abandoned pretest probability (PTP)-based strategy and recommended the referrals of CCTA in patients with angina or abnormal ECG . On the 2016 NICE guideline-determined diagnostic strategy (NICE strategy) , 13 and OPtimal Evaluation of stable chest pain to Reduce unnecessAry utilization of cardiac imaging TEsting) study is designed to compare the effectiveness and safety of two proposed diagnostic strategies, ESC and NICE strategy, in identification of low risk individual who may derive minimal benefit from CCTA among patients with SCP suggestive of CCS in a pragmatic RCT.Therefore, the OPERATE  in Beijing-Tianjin-Hebei region, which has more than 100 million population. The total annual number of patients presenting with SCP suggestive of CCS and referred to CIT in these high-volume sites is more than 30,000 and 20,000, respectively. Subjects are considered eligible for inclusion if they suffer SCP suggestive of CCS, are more than 30 years of age, and willing and able to give informed consent. Eligible subjects are clinically stable, have no history of CAD and will undergo routine electrocardiogram (ECG) and echocardiography according to the latest guideline . TrainedBaseline characteristics are defined and collected as described previously , 18, 22.After collection of baseline clinical data, eligible subjects will be randomized (in blocks of 2) sequentially 1:1 to start double-blind diagnostic strategy with 1 of 2 regimens: NICE and ESC. The computerized randomization list is generated independently by a statistician from the statistical and data coordinating group who will not participate rest of the study. Both investigators and subjects are blinded to the allocation process until the end of trial . Once the subject is ready for randomization, the corresponding envelope will be opened by the independent strategy assignment expert panel consisting of cardiologists who will not participate rest of the study. The expert panel determine whether the subject should be referred to CCTA according to the given diagnostic strategy (see below) and only send the final recommendation about referral or deferral of CCTA to the physician and subject.For a subject in high risk group according to each strategy, CCTA should be referred as the first-line CIT and other CIT, including noninvasive functional testing (NFT) and invasive coronary angiography (ICA), will be considered as an alternative. In both diagnostic strategies, subjects determined to be at low risk will be referred to optimal medication treatment (OMT) with no immediate CIT. The decisions regarding OMT will be done at discretion of the referring physicians according to recent primary prevention guidelines , 25, andAccording to NICE strategy, subjects with nonanginal chest pain and normal ECG are classified into low risk group. Subjects with typical and atypical angina or nonanginal chest pain with abnormal ECG are classified into high risk group .For each subject, ESC-PTP is calculated using age, sex and type of chest pain according to 2019 ESC guideline for the diagnosis and management of CCS  and RF-CAll participating sites will use standard equipment, procedure and interpretation for CIT, as defined by current practice guidelines , 26–29 aSpecially, CCTA images are acquired using 64-slice, or greater, multidetector CT scanners and all DICOM imaging data will be sent to the CCTA Core Laboratory  for further analysis. In the CCTA Core Laboratory, all CCTA images will be analyzed by three experienced observers, 2 radiologists and 1 cardiologist, who are blinded to the other data. In image analyses, all segments ≥ 2 mm in diameter are identified and analyzed using the updated Coronary Artery Disease–Reporting and Data System (CAD-RADS) . The perThe result of CCTA, as well as other CIT, will be provided to the local physician and he or she will make subsequent clinical decisions for the subject, such as additional CIT and clinical interventions like cardiac rehabilitation, OMT and coronary revascularization (CR), based on recommendations from clinical guidelines , 26–29 aThe primary outcome, main secondary outcomes and other outcomes are described below and all outcomes are included Table The key secondary endpoint indicating the safety of diagnostic strategy is the time to first MACE using composite of the following clinical events: All-cause death is used rather than cardiovascular death to eliminate the need for possibly difficult adjudication of causes of death, especially given the relatively low mortality; Nonfatal myocardial infarction (MI), including spontaneous and procedure-related, is defined according to the Fourth Universal Definition of Myocardial Infarction ; HospitaIn addition to the individual components of MACE, other secondary endpoints indicating the safety of diagnostic strategy include the followings: All-cause death, nonfatal MI, hospitalization due to unstable angina, overall exposure to radiation and complications (classified as major and minor) related to cardiovascular procedures which occur within 72 h. The estimated cumulative radiation exposure over the entire trial will be measured in milliSieverts using administered dose on CCTA, converted contrast agent dose for nuclear and administered radiation dose (kerma air product or dose length product) or fluoroscopy time for angiography .Another secondary endpoint indicating the effectiveness of diagnostic strategy is resource use patterns . The diagnostic metrics of CCTA during initial management, including diagnostic yield proportion of normal CCTA and necessary CCTA, will be also calculated. Necessary CCTA is defined as obstructive CAD or alteration of OMT due to nonobstructive CAD on CCTA. Other effectiveness outcomes include time length of initial management and health-related quality of life (HRQOL).The planned duration of the study is 24 months: 12 months for the enrollment period, defined as first visit of first subject to first visit of last subject and 12 months for the follow-up period. Study will preliminarily end when the following have occurred: at least 800 subjects have been randomized and 12 ± 1 month have elapsed since the last subject is randomized. All subjects will be followed for a minimum of 1 year and we will also try to continue the further follow-up after 1 year at 12-month intervals until death or withdrawal. Follow-up will be performed at 1st month (± 7 days), 3rd moth (± 14 days), 6th month (± 14 days), and 12th month (± 28 days) after randomization. The information of clinical visits is shown in Table The sample size calculation is performed using R . To compute a weighted average rate as the projected rate of CCTA without obstructive CAD for a diagnostic strategy, data from previous literatures used in the sample size calculation include rate of CCTA without obstructive CAD in high risk group and distribution of subjects in low and high risk group.For NICE strategy, theses parameters fluctuated over a wide range in different cohorts , 13, 16.For the analysis of primary endpoint, on 2-sided test 5% significance level and for the 1:1 allocation ratio, a sample size of 800  will provide more than 99% power between NICE and ESC arm. Thus, the final sample size is chosen to be 800 subjects (400 in each randomization group).All statistical analyses are performed by the statistical and data coordinating group using R  or MedCalc . Two-tailed P < 0.05 is considered statistically significant. The primary analysis is conducted according to a modified intention-to-treat (ITT) principle. All subjects who undergone randomization and don’t withdraw before assigned intervention (CCTA or no further testing) or other CIT during the initial management will be included. Categorical variables will be described by counts and proportions and compared by chi-square tests or fisher exact test. Continuous variables will be described by mean and standard deviation or median and interquartile range and compared through t test or Mann-Whitney U tests.The comparison of primary outcome between two diagnostic strategy regimens was expressed as risk ratio, along with 95% confidence interval and number needed to treat. The per-protocol analysis excluding subjects with insufficient compliance or with other major protocol deviation(s) and the traditional ITT analysis including all subjects who are randomized will be performed as prespecified sensitivity analyses. Different diagnostic metrics, such as initial CIT without positive result, unnecessary CCTA and CCTA with no sign of CAD, will also be considered as sensitivity analyses. To explore the consistency of strategy effectiveness according to specific subject characteristics, a limited number of prespecified subgroup analyses for the primary endpoint are conducted using interaction terms in log-binomial regression models. We will also undertake a hierarchical analysis  using the matched win ratio method .Kaplan-Meier curves for cumulative event-free estimates survival from the first of the following: endpoints of concern, death, the end of 1-year follow-up period or loss to follow-up, are presented graphically and the Log-rank test will be used to calculate the corresponding p. A Cox proportional hazards model will be used to compare the time to study endpoint and to account for heterogeneity among subjects, the models will be adjusted for pre-specified and minimization covariates, which are selected on the basis of their established importance in other CCS cohorts, highly complete data capture, and a sufficient range of values for risk to vary among subjects meeting study eligibility criteria.Multiple imputation using the R package Multivariate Imputation by Chained Equations algorithm will be used for missing data in ITT analysis of primary endpoint to ensure all subjects could be included in the ITT analysis, as well as HRQOL analysis . StrategRecent studies have demonstrated that performance of CIT was significantly influenced by PTP –39 and sThe risk of incorrectly deferral of CIT for patients with severe CAD should emerge as particularly strong candidates accounting for the overuse of CIT in clinical practice . In factAlthough PROMISE minimal risk tool (PMRT) has been also validated in the CICM-SCP registry and demonstrated a robust risk assessment of SCP in several other validation studies , 44–46, Several representative RCTs , 48, 49 In patients with SCP suggestive of CCS, CCTA provided excellent performance for diagnoses and prognosis of CAD in a noninvasive and feasible way  and majoIn conclusion, we anticipate that the OPERATE study will investigate the real effects of symptom-focused (NICE) and PTP-based (ESC) diagnostic strategy on decisions of clinical management and subsequent clinical outcomes in patients with SCP suggestive of CCS.Below is the link to the electronic supplementary material.Additional File 1: SPIRIT 2013 checklist


Electrospinning is an effective method for producing high-quality biopolymer nanofibers, such as cellulose and chitosan. Cellulose nanofibers have excellent mechanical properties and biocompatibility, making them a promising material for tissue engineering. Chitosan nanofibers are biodegradable, biocompatible, and antimicrobial, making them ideal for biomedical applications. The electrospinning parameters, including solution concentration, power supply voltage, orifice diameter, temperature, humidity, and flow rate, play a crucial role in determining the nanofiber diameter, morphology, and mechanical properties, as well as their suitability for various applications.This systematic review aims to synthesize and evaluate the current evidence on the influence of electrospinning parameters on the production and properties of cellulose and chitosan nanofibers.A comprehensive search of electronic databases was conducted to identify relevant studies. The inclusion criteria were studies that investigated the effect of electrospinning parameters on cellulose and chitosan nanofibers.It was found that for cellulose, the average fiber diameter increased with increasing each of solution concentration, power supply voltage, orifice diameter, temperature, and humidity. Contrary to tip - collector distance and some optimal points in temperature, where average fiber diameter decreased. For chitosan, the change in voltage and tip to collector distance did not alter the average fiber diameter except for some readings of voltage, which behaved differently. On the other hand, the average fiber diameter increased with increasing flow rate.The review highlights the importance of considering electrospinning parameters in the production of high-quality biopolymer nanofibers and provides insights into the optimization of these parameters for specific applications. This review also highlights the need for further research to better understand the underlying mechanisms of electrospinning and to optimize the process to produce biopolymer nanofibers with improved properties. They’red by Electric field created axial direction of polymeric flow, yielding spinning operation  in record. Furthermore, Boolean operators  were appropriately employed for identifying research. For all the papers included in this review, the searching words that is used for advanced search was as: “Electrospinning” OR “Biopolymers Nanofibers” [All fields] OR “Nanofibers” [All fields] OR “Electrospinning Parameters” [All fields] AND “Cellulose & Chitosan” [All fields].2.32.3.1All prospective and retrospective observational studies  articles conducted in any field of Cellulose & Chitosan and written only in English language were included if they reported the influence of electrospinning parameters on biopolymers nanofibers.2.3.2Those studies did not report our variables of interest or comprised incomplete information . Articles where the full text cannot be accessed were excluded from this systematic review.2.3.3The quality of each article was evaluated by using a 14-points checklist. ‘High quality’ for an article with score > and = 70%. A score of 69 to 51% considered as “moderate quality”, less than or equal to 50% were considered “Poor quality”. Two authors scored each article individually and the mean value of the results was used. No article got excluded due to inferior quality, as all articles scored more than 50%.2.4Using Microsoft Excel 2019, authors created a data extraction tool to collect all the needed data from selected articles. Data related to characteristics of the articles such Parameter, Material, Methods and Preparations, Results, Findings were extracted. Solution concentration and applied volt were also extracted. The data extraction was carried out by two authors independently. When disagreement was encountered by the two authors, a third author was delegated to extract the data. Microsoft Excel 2019 was used to analyze the Effect of the parameters and the results were depicted in charts and tables.2.5This study design was made to determine the risk of bias of individual. Validity of the data were assured for each outcome by identifying the methods and analyses that were employed to assess the outcomes. Both study and outcome level information were used in synthesis.2.6As the effectiveness was presented in percentage without always mentioning the means and confidence intervals, the principal summary measures could not be estimated.2.7After assessing the individual study, the bias across the studies was evaluated. The risks of bias included studies from publication. Selection and reporting are described in the discussion section of the review.33.1During our advanced research, we identified 11,450 articles, and we used the PRISMA flow diagram method  to demon3.2The electrospinning technique was initially developed by Rayleigh in 1897 and presented in detail by Zeleny in early 1900 . This wh3.3The electrospinning process is determined by several parameters. They are divided into three categories: machine parameters, material parameters, and ambient parameters. Applied electric field, distance between needle and collector, flow rate, and needle diameter are machine parameters. Solvent type, polymer concentration, viscosity, and material conductivity are material parameters. Relative humidity and temperature are ambient parameters. All these parameters have an impact on the production of smooth, bead-free electrospinning fibers, therefore, it is important to fully understand the impacts of all of these key parameters to obtain a better knowledge of the electrospinning process and production of polymeric nanofibers ,25.44.1The electrospinning method is based on the phenomenon of a charged jet extending uniaxially. Changing the concentration of the polymeric solution has a tremendous impact on the stretching of the charged jet. For example, when the concentration of the polymeric solution is low, the applied electric field and surface tension cause the entangled polymer chains to break up before reaching the collector. Beads or beaded nanofibers are formed because of these fragments; therefore, at very low concentrations, the average fiber diameter is high until the critical value of concentration, as explained in 4.24.2.1Fiber diameter increases with the increase of the applied voltage, as depicted in 4.2.2Increasing the flow rate above a critical threshold causes an increase in fiber diameter, as in 4.2.3The needle to collector distance affects deposition time, evaporation rate, and whipping or instability interval so, the nanofiber morphology depends on it. To create smooth and uniform electrospinning nanofibers, a critical distance must be maintained, as explained in 4.2.4The average fiber diameter increases due to the increase in the orifice diameter, see 4.3Environmental (ambient) variables such as relative humidity and temperature, in addition to machine and material characteristics, have recently been shown to impact the diameter and shape of nanofibers. The average diameter increased by increasing humidity, see 5Electrospinning is a reliable and widely used method that has been implemented in various fields. Cellulose and chitosan nanofibers are particularly popular due to their numerous applications. Cellulose nanofibers, for instance, find use in tissue engineering and medical implants, while chitosan nanofibers are used for water purification and air filters. To improve the characteristics of electrospinning nanofibers, extensive research has been conducted to explore the impact of various parameters such as concentration, viscosity, type of solvent, surface tension, conductivity, applied voltage, flow rate, TCD, orifice diameter, humidity, and temperature. Based on the findings, it is concluded for:•The average fiber diameter increased gradually with the increase in solution concentration.•With the increase of the applied voltage, the average fiber diameter increased.•Due to the increase in TCD, the average fiber diameter decreased gradually.•The average fiber diameter increased with the increase of the orifice diameter.•With the increase in spinning and solution temperature, the average fiber diameter gradually increased.•the average fiber diameter increased with the increase in humidity.Cellulose:•The average fiber diameter was almost constant with the increase in the applied voltage.•The average diameter gradually increased with the increase of the flow rate.•The average fiber diameter is nearly fixed by the increase of the TCD.Chitosan:According to previous findings, it was discovered that to produce fine fibers from cellulose and chitosan, optimized monitoring of the different parameters  should be performed, and this will be accomplished through the study of changing these parameters all at once rather than individually.All authors listed have significantly contributed to the development and the writing of this article.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.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


We are delighted to publish the second volume of the Research Topic: Antimycobacterial Drug Discovery—Molecular Therapeutics and Target Identification. The Research Topic continues the discussion with an emphasis on the identification of novel antimycobacterial compounds and, more broadly, antibacterial compounds isolated from natural products or derived synthetically. Studies on the mechanisms of action of these bioactive molecules provide insights into the design of safer therapeutics and antimicrobials for the treatment of multidrug-resistant infections.Khare et al. The authors provide a comprehensive summary of the effects produced by alkaloids, phenolic compounds, terpenes, and coumarins against drug-resistant pathogens, with a focus on the major determinants of antibiotic resistance, such as efflux pumps, cell permeability, and modification of the antibiotic targets. Rubio et al. report on the mechanism of cellular uptake of mycolactone, a lipid toxin produced by Mycobacterium ulcerans, from the circulating plasma Using a combination of dynamic light scattering and fluorescence spectroscopy, the authors demonstrate how mycolactone behaves in solution and how it interacts with serum albumin and lipoproteins in plasma. Monitoring plasma levels of complexes formed by mycolactone and albumin/lipoproteins could be useful for the early diagnosis of patients with Buruli ulcer disease. Wang et al. investigate the effect of the labdane diterpenoid andrographolide on the hemolytic activity of S. aureus. Andrographolide dose-dependently inhibits hemolytic activity by significantly downregulating the transcript levels of the Hla gene, which in turn is responsible for the expression of the crucial virulence protein Hla. Molecular dynamics simulations of Hla-andrographolide interactions provide valuable insights into the structural requirements for the observed activity and have the potential to design more potent andrographolide analogs. Hoffmann et al. demonstrate the repurposing potential of nitroxoline for the treatment of multidrug-resistant tuberculosis (MDR-TB). Originally approved for the treatment of urinary tract infections, nitroxoline was tested in a panel of MDR-TB cells and found to have MIC values of 4–8 mg/L. Finally, Korycka-Machala et al. report the synthesis of thiosemicarbazide-based compounds and their structure-activity relationship against Mycobacterium tuberculosis. The authors demonstrate the efficacy of these compounds in inhibiting the growth of intracellular M. tb and reducing biofilm formation. The selection of resistant mutants and their whole genome sequencing identified the mmpR5 gene as a target for these analogs.The second volume begins with a review of the antimicrobial properties of phytochemicals by Taken together, the second volume of this Research Topic: “Antimycobacterial Drug Discovery: Molecular Therapeutics and Target Identification”, provides recent advances in the development of antibacterial and antimycobacterial agents. We acknowledge the contributions of all the authors that made this research volume possible.


With an increasing number of new scientific papers being released, it becomes harder for researchers to be aware of recent articles in their field of study. Accurately classifying papers is a first step in the direction of personalized catering and easy access to research of interest. The field of Density Functional Theory (DFT) in particular is a good example of a methodology used in very different studies, and interconnected disciplines, which has a very strong community publishing many research articles. We devise a new unsupervised method for classifying publications, based on topic modeling, and use a DFT-related selection of documents as a use case. We first create topics from word analysis and clustering of the abstracts from the publications, then attribute each publication/paper to a topic based on word similarity. We then make interesting observations by analyzing connections between the topics and publishers, journals, country or year of publication. The proposed approach is general, and can be applied to analyze publication and citation trends in other areas of study, beyond the field of Density Function Theory. Basically, this change is driven by the fact that the institute that introduced this approach  has decided to discontinue its support, primarily due to the huge cost of administration and paper handling during manuscript submission. Therefore, a more dynamical and flexible method is necessary. A method were different topics capture the continuous change in the scientific development and scientific interest and are able to map reviewers and even journal in this arena. In this respect, the use of topic modeling from machine learning is gaining a lot of attention3. In this paper, we propose an unsupervised learning technique that focuses on finding text correlations and thus classify documents according to a small set of topics. These topics are found through an unsupervised machine learning technique that creates clusters of meaningful words in the document selection, by finding similarity between them in a latent space. Though the idea of topic modeling has been used in the past to classify research topics5, in this work, we start from a well-defined database, and apply this methodology on publications related to the use of electronic structure packages.For many years, scientific documents in basic sciences were classified using the Physics and Astronomy Classification Scheme (PACS) method, which maps a series of codes with keywords to cover the main topics of the publication. This is usually done by the author, and provides relative freedom for the actual classification. Though this is still one of the methods used by several journals, this step is transitioning into a more focused type of topic classification6. Density Functional Theory (DFT) is the most used methodology to characterize materials at the atomic scale8. It falls into the so called electronic structure methods and it has the benefit that a large number of computational codes and software employ this method, which have created communities around them, supporting the research efforts. We base our document selection on research papers citing those computational code libraries, in order to reduce our scope to only DFT-related papers.While a general method to categorize topics in basic sciences would be ideal, in this paper, we have decided to focus in a particular research field, where we can have access to a very well controlled database10. However, our dataset downloaded from the Web of Science database, does not keep records of the PACS numbers and only retains keywords. Moreover, as DFT is a methodology with many different applications, in fields other than physics, not all papers follow the PACS structure. This shows again, the necessity for a new classification system, based purely on the content of the documents. Our approach is not only able to classify documents on content but also can be used to find novel topics and relationships between topics which can be used to identify new and emerging research fields.Some efforts to analyze DFT-related papers from different subfields have been made, but they classify the articles based on the PACS numbers6, through the Web of Science database (WoS), but expanded to include articles published in 2020.The research papers and their abstracts were obtained using the same methodology as in previous workOnce we extracted the abstracts from the WoS database, we performed several pre-processing steps to capture only the most meaningful words out of the text. First, we deleted certain groups of words, such as “left arrow” or “vertical bar,” that are only used to create mathematical representations. For the same reason, we removed all characters between two dollar signs, as this is a standard command to make equations, which would only confuse the algorithm. There is one exception to this rule if an underscore is found between the two dollar signs, as that pattern is used for chemical formulas that are essential and meaningful notions in sub-fields of DFT. We also remove publishing and copyright tags, DOI numbers, and all non-alphanumeric characters.11 list of stopwords and added others based on our own experience with the data after rounds of trial and feedback. For example, “theory” and “method” are not specific to some areas of physics like magnetic properties or gas absorption. We also remove all single letters and digits, often used in mathematical expressions or to quantify a value and don’t hold meaning intrinsically. We then create bigrams and trigrams, groups of two or three tokens that often appear one after the other and will now be linked as one word by an underscore. Examples would be “ab initio” and “density functional theory”. Finally, the tokens are lemmatized to reduce words to their root, and the resulting list of tokens is filtered to only keep the ones that are in at least two documents and in less than 20% of the papers. From this final list are created a dictionary and a Bag of Words (BoW) which are the input to our algorithm.Then, the leftover sentences get tokenized, which means the text is broken down into units, creating a list of tokens from the text. From those lists, we take out all stopwords, or words that are so common they do not help differentiate different sub-fields. We used the standard NLTK12 is a topic modeling algorithm which uses Bayesian statistical methods to generate text, from documents passed in as input in the BoW. This means that LDA does not take into account the order of words. In fact, an LDA model creates a set of topics where each topic is a probability distribution over a corpus of words drawn from the distribution Latent Dirichlet Allocation (LDA)M as the total number of documents, N as the total number of words in a document m, and k as the number of topics. As shown in Fig. Here, we define 13 that already has a handy set of functions to create, evaluate and visualize LDA models. We used two bag of words (BOWs): one with all documents in our dataset from 1990 to 2019, and one with only documents published in 2020. The 2020 corpus was used as a test set.For topic modeling, we implemented a Latent Dirichlet Allocation (LDA) algorithm with the Python library Gensimk), from 5 to 80, while keeping all other parameters null. For this analysis, we chose to tune the parameters left for 15, 25, 35 and 45 topics. All models were evaluated with the 14, which is the coherence score with the strongest correlation to human rankings. We then ran 480 models to tune hyperparameters including k. For each value of k, the model that gives the highest We created 16 models with a different number of topics Here, we describe our dataset, experiments and results.6.To create a dataset of research papers focused on DFT, we used the graphical interface “Web of Science” (WOS) and downloaded all articles citing some of the most commonly used computational packages to study crystalline systems. We acquired all papers from the first citation up to 2019 for our training set; and all citations in the 2020 year for our test setFigure k = 15 and k = 45 seem to be somewhat well distributed, as no topics is contained in more than 18% and 11% of the documents. This can easily be deduced via the y-axis of the histograms, that are also a quick way to get a general idea of a distribution: if the y-axis range is larger, it means that at least one topic takes a bigger part of the documents, which means the distribution is less uniform. Similarly, we can study the range of the attributed normalized number of documents. The model with 25 topics has the bigger range at 0.255, followed by 15 topics at 0.175 and 45 topics at 0.105. The smallest range, and hence the model that has a more uniform distribution is for k = 35 with 0.086. Indeed, to get 90% of the documents, it takes the first 8 topics (or 53% of the topics) with the highest proportion for k = 15, 24% of the topics for k = 25, 57% for k = 35 and 37% for k = 45. Moreover, the model with 35 topics is also the one that has the highest minimum percentage of documents.We can see that the model with 25 topics is extremely unbalanced, with only 4 topics accounting for up to 80% of the documents. Next, the LDA models for k = 35 and k = 45 also seems to imply that 15 topics is not enough for our corpus. In fact, we find evidence for topics specialization that further confirm the benefits of a higher number of topics. For example, in the k = 15 model, Topic 3 contains the following words: “formation”, “activity”, “hydrogen”, “metal”, “reaction”, “mechanism”, “process”, “catalyst”, “oxidation”, and “li”. We can map this topic to two different topics in the k = 35 model: it shares the words “activity”, “metal”, “reaction”, “catalyst”, “oxidation” with Topic 6 and the words “reaction”, “formation”, “mechanism”, “process” with Topic 27. We can distinctly see that it splits into two categories, one is more focused on specific processes and applications (Topic 6) and one is more general (Topic 27).The diversity in the models for k = 35. For these two reasons, we will mostly focus on this particular LDA model and its results in the rest of this section.We can conclude from these analyses that the most balanced model is for Figure 16. Figure We can also analyze the topics of our model by visualizing them with the t-SNE reduction methodFigure We computed, for each topic independently, the normalized and weighted number of documents per year. Every year, there is an exponential amount of DFT-related articles being published, and consequently, almost all topics also grew exponentially. To compare them, we calculated the power law factor of each curve since 2000. Even though some topics’ evolution are better fitted with an exponential curve, the power law function yields the highest mean A total of nine topics have a power factor lower than 1 and while they still grew in importance since 2000, a few of them are starting to stabilize and even lose some interest from the DFT community. For example, Topic 18 on DFT schemes and Topic 19 on nanotubes, which have the lowest factors recorded, have not gained any new momentum in the recent years and Topic 18 also observed a decrease of publications in 2017 for the first time ever. On the contrary, Topics 24 related to electrochemical processes and lithium batteries, as well as Topic 4 , Topic 6  and Topic 31 (development of new methods) are growing at a significantly high rate and seem to be among the most important subjects in the field of DFT today. Other subjects are all developing at intermediate rates, representing the diversity of DFT and how important the methodology is for different topics.We report in Fig. Figure It is interesting to compare the evolution of Topics 14 and Topic 31, as they are related to each other. Topic 14 contains words such as “data”, “model” and “simulation” which represent the community’s effort to incorporate code, machine learning and new computing methods into their work. This topic has been growing steadily over the years, as it becomes a bigger part of today’s experiments. Secondly, Topic 31 relating to the improvement and optimization of code and processes, only started gaining momentum in 2014, about 10 years after Topic 14. However, its growth was faster, which places both of the topics at the top of the most published subjects in 2020 in DFT. We can also mention the evolution of Topic 4 related to graphene and monolayers. It started to grow a few years after 2010, which is the year that the Nobel Prize in physics was awarded to Andre Geim and Kostya Novoselov for their work regarding the two-dimensional material graphene.Looking at the number of citations per year, per topic in Fig. Hence, looking at the trends of citations over the years might give us more insight and more predictive value than only the number of published papers. For topics that are only starting to attract more interest, like Topics 6 and 24, we can trace back the beginning of the trend to the late 2000s or early 2010s whereas topics that have been in place for longer such as Topics 8, 10 or 14 can be attributed to discoveries in the late 1990s.We also studied the spatial evolution of topics in DFT over the years, especially, by countries. To do so, we identify countries using the affiliations of all authors. However, we only count a country once for each paper.Figure We can accurately predict that China will soon dominate the field of DFT as its number of publications will only keep growing. Figure Similarly to Fig. The 15 journals displayed have the highest number of publications and are arranged in order from left to right. Even though some journals like Surface Science are specializing in one topic, and sticking with it over time, we can notice how most journals are present in several different subjects. Two key journals, Physical Review B and Journal of Physical Chemistry C are both influential in DFT and were expected to be the largest contributors to the field. Moreover, as DFT is mostly used in physics and chemistry, it is not surprising that those two journals both focused on condensed matter analysis each represent one of the two domains.In fact, the three journals in positions 2, 3 and 4 are related to the chemistry side of DFT. Even though, they have similar targets, they evenly distribute the publications between them. The Journal of Chemical Physics focuses on Topic 14 and 18, Journal of Physical Chemistry C contributes more to Topics 5, 6, 8 and a few others while Physical Chemistry Chemical Physics has a more diverse range of subjects. Hence, it seems that the Journal of Chemical Physics contributes more to methodologies and not material characteristics, judging by the words present in Topics 14 and 18 (modeling/approximations). Even though physics researchers mostly publish their articles in one particular journal, chemists have more options available depending on their interests.Figure Figure Bibliometrics and topic modeling are powerful tools that can be used to study and evaluate the evolution and impact of any given field. In this paper, we developed several analyses related to the evolution of topics with emphasis on the specific field of DFT. Here, evolution was considered in terms of time (over the years), and space (across countries), and also with respect to journals, publishers, institutions, and subjects based on information from over 120,000 papers in Density Functional Theory. We find that automatically classifying papers based on their abstract is a promising option for improved, faster, and more objective categorization. In fact, most of the 35 topics resulting from our LDA model were consistent with our document corpus and could be attributed to a name.Even though most packages are developed in Europe, they are mostly used in the USA and China, that are dominating the amount of publications created in the DFT field. While the two countries share interests in a lot of different subjects, American scientists work with many of the most important DFT packages, and researchers in China use mostly VASP, which is a licensed package. This difference might come from the intrinsic set of tools each software has, and the subfields they serve the most. Indeed, China seems to direct most of its work to materials science and applications as they focus on 2D materials (Topic 4), chemical reactions (Topic 6) or batteries (Topic 24) while researchers in the USA published most of their work in simulations (Topic 14) and new methodologies (Topic 31).Moreover, two of the most recurrent topics in our analysis, and that receive the most interest from influential journals and countries are related to code development, simulations and modeling. As our work focuses on computational packages, it is not surprising that some papers focus on the methods and processes of software. However, the large amount of interest in the subjects suggests a recent and strong effort in the physics and chemistry fields to develop and optimize code packages and libraries.The topic modeling done in this article was created using the abstracts of papers and already resulted in coherent and clear topics. Future work could include replicating our methodology on the full-text of all 120,000 papers in our dataset. Further, the approach used is quite general, and can easily be adapted to apply to other fields of study, beyond DFT.


Antimicrobial resistance (AMR) surveillance plays an important role in early detection of resistant strains of pathogens and informs treatments decisions at local, regional and national levels. In 2017, Tanzania developed a One Health AMR Surveillance Framework to guide establishment of AMR surveillance systems in the human and animal sectors.We reviewed AMR surveillance studies in Tanzania to document progress towards establishing an AMR surveillance system and determine effective strengthening strategies.We conducted a literature review on AMR studies conducted in Tanzania by searching Google Scholar, PubMed, and the websites of the Tanzania Ministry of Health and the World Health Organization for articles written in English and published from January 2012 to March 2021 using relevant search terms. Additionally, we reviewed applicable guidelines, plans, and reports from the Tanzanian Ministry of Health.We reviewed 10 articles on AMR in Tanzania, where studies were conducted at hospitals in seven of Tanzania’s 26 regions between 2012 and 2019. Nine AMR sentinel sites had been established, and there was suitable and clear coordination under ‘One Health’. However, sharing of surveillance data between sectors had yet to be strengthened. Most studies documented high resistance rates of Gram-negative bacteria to third-generation cephalosporins. There were few laboratory staff who were well trained on AMR.Important progress has been made in establishing a useful, reliable AMR surveillance system. Challenges include a need to develop, implement and build investment case studies for the sustainability of AMR surveillance in Tanzania and ensure proper use of third-generation cephalosporins.This article adds to the knowledge base of AMR trends in Tanzania and progress made in the implementation of AMR surveillance in human health sector as a contribution to the global AMR initiatives to reduce AMR burden worldwide. It has highlighted key gaps that need policy and implementation level attention. Subsequently, a holistic One Health AMR Surveillance Framework was developed to guide the establishment of AMR surveillance systems in the human, animal and environmental health sectors. The country is bordered by more than eight countries, which poses a high risk of pathogen importation into the country. In addition, several socioeconomic, demographic and environmental factors also facilitate the emergence and spread of microorganisms; thus, robust health systems are paramount for detecting, responding, and mitigating the effects of the resistant microbes.6 In fact, recent studies have shown the rampant use of antibiotics by most COVID-19 patients without bacterial co-infections.7In this current global coronavirus disease 2019 (COVID-19) pandemic situation, where scientists are struggling to find an effective treatment for COVID-19, antibiotics have been widely used to manage COVID-19, either to treat COVID-19 itself or co-infections.This paper reviewed AMR surveillance studies and documents the progress made in establishing the AMR surveillance system in the human health sector in Tanzania and provides recommendations for strengthening it. The literature review was essential to contextualise the AMR situation in the past decade and the need to strengthen AMR surveillance.We searched Google Scholar, PubMed, and websites of the Tanzania Ministry of Health and WHO written in English and published from January 2012 to March 2021. We used the search terms: ‘antimicrobial resistance’, ‘bacterial resistance’, ‘antibiotic resistance’; ‘AMR surveillance’, or ‘surveillance’ or ‘cross-section’ or ‘review’; and ‘Tanzania’. All words were searched together, and, in some instances, two of the three words were used. We reviewed guidelines, plans, and reports from the Ministry of Health to describe the Tanzania AMR surveillance system’s objective, surveillance sites, data collection, reporting, analysis, interpretation, and dissemination, coordination of AMR surveillance activities, and funding of AMR surveillance.8 Tanzania Mainland has 26 administrative regions, 139 districts and 184 councils. The council divides into divisions, then wards, and streets/villages. The local government authorities (or councils) are the most important administrative and implementation units for public services.The United Republic of Tanzania comprises Tanzania’s Mainland and the semiautonomous Islands of Zanzibar, and it lies on the East African coast. The Tanzania 2012 population census was 44 928 923.9 Dispensaries provide preventive and curative out-patient services. In contrast, health centres admit patients and sometimes provide surgical services. Council hospitals provide healthcare to referred patients and provide medical and basic surgical services. Regional Referral Hospitals provide specialist medical care. Zonal and national hospitals offer advanced  medical care and are teaching hospitals for medical, paramedical, and nursing training.9 Public, private and faith-based organisations health facilities, private pharmacies, and accredited drug dispensing outlets provide pharmaceutical services.11Health services in Tanzania are decentralised into three categories: district (primary level), regional (secondary level), and zonal and national hospital (tertiary level). The district level provides primary health care services through dispensaries at the village level, health centres at the ward level, and Level 1 Hospital at the council level.12 The most frequently isolated microorganisms from blood were Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli; and from pus, Pseudomonas aeruginosa , tetracycline (34.9%), cotrimoxazole (26.5%) and ceftriaxone (11.1%).18 Prevalence of methicillin-resistant S. aureus decreased from 41.2% in 2013 to 9.5% in 2015, but rose to 66.7% in 2018.15K. pneumoniae was resistant to ampicillin (100%), cotrimoxazole (96.3%), ceftriaxone (95.7%), amoxicillin/clavulanate (94.6%), ceftazidime (90.9%), gentamycin (86.4%) and cefepime (75.6%).19 Compared to other Gram-negatives, E. coli was more resistant to ampicillin, amoxicillin-clavulanic acid, gentamycin, tetracycline, ciprofloxacin, amikacin, third-generation cephalosporins (ceftazidime and ceftriaxone) and cefepime.,13,15,16,19,20 Notably, P. aeruginosa was resistant to cefepime (93.8%).13 Overall, most studies documented high resistance rates of Gram-negative bacteria to third-generation cephalosporins.22A total of 10 articles on AMR in Tanzania were retrieved and reviewed. These studies were conducted at either the zonal or regional referral hospitals between 2012 and 2019. Four of the 10 studies were conducted at Kilimanjaro Christian Medical Centre in the Kilimanjaro region, three at Bugando Medical Centre in Mwanza region, two at Muhimbili National Hospital in Dar es Salaam region, and one study each at Maweni Regional Referral Hospital in Kigoma region, Musoma Regional Referral Hospital in Mara region, Sumbawanga Regional Referral Hospital in Rukwa region, St. Benedict Ndanda Hospital in Mtwara region, Bagamoyo District Hospital in Pwani region, Sekou Toure Regional Referral Hospital, Nyamagana District Hospital, and Sengerema District Designated Hospital in Mwanza region . Blood, ruginosa . S. aure23 The objectives of AMR surveillance are to routinely collect, analyse, and interpret quality AMR data to generate evidence on trends and the burden of the WHO priority pathogens, and monitor AMR interventions’ effectiveness. The country also established a national Multi-Sectoral Coordinating Committee (MCC) to oversee and coordinate all AMR-related activities in all sectors. The Chief Medical Officer of the Ministry of Health and the Director of Veterinary Services, Ministry of Livestock and Fisheries alternate as co-chair of the committee. The committee is composed of representatives from the human, animal and environmental health sectors, as well as livestock and food production stakeholders, and those from medical and agricultural universities. The WHO, Food and Agriculture Organization, United States Centres for Disease Control and Prevention, Management Science for Health and World Organization for Animal Health, are also represented in the MCC. There are designated national AMR focal points from animal and human sectors that form part of the MCC secretariat, as well as four multisectoral Technical Working Groups on (1) awareness, effective communication and education; (2) knowledge, surveillance, research and sustainable investments; (3) sanitation, hygiene and infection prevention and control; and (4) antimicrobial use stewardship. The MCC and surveillance TWG meets at least once every quarter of the year. The whole coordination structure operates under the ‘One Health’ and whole-of-government approach.In 2018, Tanzania took the first step of developing a One Health National AMR surveillance framework to guide the establishment of AMR surveillance programmes in the country. The framework provides a holistic approach to monitor trends of infections and resistance that will inform standard treatment guidelines that support best practices for patient care, and links AMR information from the human, animal and environmental health sectors.25 In the first phase of the national Tanzania AMR surveillance there were two laboratory levels: coordinating laboratory and site (sentinel/participating) laboratories. As of 2022, there are a total of nine AMR sentinel sites which are active and functional. The sentinel sites include Muhimbili National Hospital, Mbeya Zonal Referral Hospital, Bugando Medical Centre, Kilimanjaro Christian Medical Centre, Mnazi Mmoja Hospital in Zanzibar, Temeke Regional Referral Hospital in Dar es Salaam region, Morogoro Regional Referral Hospital in Morogoro region, Maweni Regional Referral Hospital in Kigoma region, and Benjamin Mkapa Hospital in Dodoma region  or non-susceptible, which include intermediate (I) and resistant (R) according to clinical breakpoints defined by Clinical and Laboratory Standards Institute. Patient clinical data, including infection origin , age, gender and admission types  are collected regardless of culture positivity or negativity. Infection origin are categorised as hospital-acquired (specimen from an in-patient admitted for > 2 days) or community-acquired (specimen from an out-patient or in-patient admitted for ≤ 2 days).23Tanzania started with laboratory-based AMR surveillance in healthcare settings, as laboratory-based surveillance is the most efficient AMR burden determination method.a region . The Nat27 However, WHONET intentionally provides only a solution for basic laboratory specimen management and result reporting and does not have comprehensive LIMS functionality.27 Laboratory departments communicate AST results immediately to clinicians as well as the infection prevention and control and AMR teams for appropriate treatment and control programs in the local setting. Target pathogens for monitoring and reporting as per the national and WHO priorities include E. coli, K. pneumoniae, Acinetobacter baumannii, S. aureus, Neisseria meningitidis, Streptococcus pneumoniae, Salmonella spp., Shigella spp., Pseudomonas spp. and Neisseria gonorrhoeae.Clinical data and AST positive culture results are recorded in the reporting forms and entered into the laboratory information system and the World Health Organization Network (WHONET), a freely available system for capturing, analysing and sharing AMR data in a standardised format. Data import into WHONET can be semi-automated using the add-on Baclink software , which allows for import from other data sources, for example, text files exported from a laboratory information management system (LIMS) or directly from a laboratory instrument.The participating laboratories must clean, collate, analyse, and create site-specific bacterial antibiograms every month. In addition, annual AMR surveillance reports are shared with the relevant clinical departments and hospital committees to increase hospital and community AMR awareness, inform treatment policies at the health facility, and encourage continued participation in the surveillance system.Antimicrobial resistance data from surveillance sites are centrally stored and managed at the NHL. The NHL conducts data quality checks, analysis, and visualisation, generates official AMR reports, and provides long-term data storage. Antimicrobial resistance surveillance reports, including trends and resistant pathogen prevalences, are generated at least twice a year and disseminated to stakeholders after approval by the AMR Surveillance Technical Working Group and the national MCC. At the same time, the clean AMR data set is transmitted to the GLASS . The AMRE. coli, K. pneumoniae, A. baumannii, S. aureus, N. meningitidis, S. pneumoniae and Salmonella spp. As per the AMR surveillance framework, all isolates are to be stored at −70 °C for future studies. Isolates with unusual, unexpected, or indeterminate resistance patterns are sent to the NHL for confirmatory testing and AST. Also, every 10th isolate from each site is sent to NHL for quality assessment.All nine sentinel sites participate in the external quality assurance which is being supported by NHL and African Society for Laboratory Medicine under the project called External Quality Assessment for Africa. External quality assurance is done twice a year for all sites and involves most of the GLASS priority pathogens, including Funds to run the nine AMR surveillance sentinel laboratories are contributed by the Government of the United Republic of Tanzania, the Fleming Fund, and the United States Agency for International Development fund under the Infectious Disease Diagnostic and Surveillance project. Therefore, there is a need to establish a sustainable funding mechanism for AMR activities in Tanzania.S. aureus, K. pneumoniae and E. coli; from urine, E. coli; and from pus, P. aeruginosa. Most studies documented high resistance rates of Gram-negative bacteria to third-generation cephalosporins. Importantly, significant progress has been made in establishing AMR surveillance; nine sentinel sites across Tanzania have been established and are generating data and there are suitable and clear coordination structures and platforms for multisectoral engagement and collaboration under One Health. However, sharing of surveillance data between sectors is yet to be strengthened. There are also few laboratory staff well trained on AMR practices.This review has shown that the most frequently isolated microorganisms from blood were 28 and this may explain the observed third-generation cephalosporins resistance trends. If this trend continues, clinicians will resort to broad-spectrum antibiotics, such as carbapenem, which are the last resort according to the Tanzania treatment guideline.30 If such a situation occurs, effective, quality, and affordable healthcare provisions, the core fundamentals for universal health coverage, will be far from being realised.Studies in Tanzania reveal increasing bacterial resistance to third-generation cephalosporins. In Tanzania, ceftriaxone is reportedly prescribed excessively and inappropriately in hospital settings,31 The AMR sentinel surveillance sites are representative and provide AMR trends and burden data. AMR surveillance system has helped to standardise routine microbiological cultures and AST in hospitals, particularly those participating in AMR surveillance according to global standards . However, the facilities still face challenges while implementing AMR surveillance, including a lack of interoperability between the sentinel site laboratory information system and WHONET to enable automatic data transfer between the two systems. The double entry of the same information in two different systems exhausts and overworks the laboratory staff. A lack of fit-for-purpose LIMS and open-source LIMS software with technical standards and functionality for AMR surveillance is a particular concern in low- and middle-income countries.27 Although WHONET is a functional and useful repository for microbiological data with capabilities for standardised data sharing, it lacks full LIMS functionality.27 Thus, there is an urgent need for investment in laboratory information technology infrastructure and data management systems that can capture high-quality laboratory and patient management data.Progress made in establishing the AMR surveillance system in the country is commendable. A total of nine sentinel sites have been established. There are suitable coordination structures and platforms for multisectoral engagement and collaboration under the ‘One Health’.33 However, despite the challenges, AMR surveillance is still ongoing in the country.There are few well-trained laboratory staff at the sentinel sites for data analysis and the production of antibiograms, which can be shared with the clinicians and AMR teams to inform on the appropriate treatment and measures to tackle AMR at the hospital or community. At the surveillance sites, frequent stock out of AST reagents and analysis, inadequate resources, and poor laboratory infrastructure for phenotypic and genotypic analysis is commonplace. Although AMR surveillance receives much support from the government as per human resources and infrastructure, there are also funds from donors. A sustainability plan is essential to prevent over-dependence on donors over time. These challenges are unique in Tanzania and have also been reported elsewhere in Africa.34 necessitating a comprehensive and coordinated AMR surveillance system that can continuously share AMR data between sectors to inform public health interventions.Sharing of surveillance data between sectors is yet to be strengthened. Antimicrobial resistance is a broad and complex issue affecting the animal, human and environmental sectors; thus, a multisectoral and multidisciplinary combat approach is needed. Antimicrobials are also widely used in animals for treatment and growth promoters. In addition, evidence suggests that antimicrobial use in animals contributes significantly to the development of AMR in humans,This study has some limitations. The review was based on reports only. We did not seek additional inputs and insights from AMR stakeholders through a standardised questionnaire or interview. As such some comprehensive views and perspectives may have been missed out. The AMR trends in Tanzania presented here should be interpreted with caution as the review was only based on 10 surveillance articles.Tanzania is currently implementing AMR surveillance in nine hospitals, and reporting of AMR data to GLASS has commenced. There are well-established AMR coordination mechanisms at health facilities and national levels to effectively implement and utilise the AMR information. Although there are challenges affecting implementation, the current AMR surveillance system in place is useful, reliable and capable of better performance. There is a need to develop, implement and build an investment case study for the sustainability of AMR surveillance in Tanzania. We recommend that the government creates a fit-to-purpose laboratory information system with functionality able to link with other systems; develops a mechanism for sustainable financing for laboratory infrastructure development and continuous supply of reagents, commodities, and laboratory materials; and invests hugely in building human capacities for bacterial identification and AST and data analysis, interpretation and utilisation.


Analytical and spectroscopic techniques were used to clarify the structural composition of the new chelates. In addition, all chelates were tested against bacterial strains and the HepG2 cell line to determine their antiseptic and carcinogenic properties. The Ni(II) complex was preferable to the other chelates. Molecular optimization revealed that H2L had the highest reactivity, followed by Hg-chelate, Ag-chelate, Ni-chelate, and Cu-chelate. Moreover, molecular docking was investigated against two different proteins: the ribosyltransferase enzyme (code: 3GEY) and the EGFR tyrosine kinase receptor (code: 1m17).Four new complexes (Ni The antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi was tested for three nickel(II) thiosemicarbazone  complexes, where L1H is (E)-4-(4-fluorophenyl)-1-(1-hydroxypropan-2-ylidene) thiosemicarbazide, L2H is (E)-4-(4-chlorophenyl)-1-(1-hydroxypropan-2-ylidene) thiosemicarbazide, and L3H is (E)-4-(4-chlorophenyl)-1-(1-hydroxypropan-2-ylidene) thiosemicarbazide; the antituberculosis activity was also tested on all compounds. These findings demonstrated the potential of Ni2+ chelates for developing antibacterial and antitubercular chemotherapeutic drugs [3)2]NO3  was premeditated against the normal straining of two Gram-positive  and two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) microbes. The antibacterial activity of the Ag(I) complex is enhanced concerning the free ligand +, 112 for [C6H5Cl-H]+, 127 for [C6H5NCl-2H]+, 201 for [C7H9N3SCl]+, 228.5 for [C8H9N3SOCl-H]+, 259 for [C9H10N4OSCl-2H]+, and 336 for [C15H15N4OSCl] , respectively (The mass spectra of Ni(II), Cu(II), Ag(I), and Hg(II) complexes shows molecular ion peaks at ectively .The interpretation of the mass spectra of the complexes Cu(II) and Ag(I) has been clarified, revealing several significant fragments. The data are in a good agreement with the proposed molecular formulas for complexes Cu(II) and Ag(I) , and this verifies the complexes’ chemical structures.m/z at 732 (8.52%) corresponding to C30H29Cl2CuN8O2S2. The various fragments of this complex provide the peaks with different intensities at different m/z positions at 348.91 (100.00%) (C15H15ClCuN3OS), 620.71 (11.20%) (C25H27ClCuN7O2S2), 585.03 (19.20%) (C22H22Cl2CuN6OS2), 578.58 (22.59%) (C24H29ClCuN7O2S), and 91.12 (17.94%) (C6H5N).The mass spectra of the Cu(II) complex shows the peak attributed to the molecular ion peak [M+] m/z at 618 (9.22%) corresponding to C15H19Ag2ClN4O3S2. The various fragments of this complex provide the peaks with different intensities at different m/z positions as at 127.57 (100.00%) (C6H6ClN), 600.64 (13.20%) (C15H17Ag2ClN4O2S2), 347.19 (9.20%) (C10H15AgN4OS), 90.15 (14.59%) (C2H6N2S), and 75.09 (8.94%) (C2H7N2O).The mass spectra of the Ag(I) complex displays a peak recognizable to the molecular ion peak [M+] 2L and Ni2+, Cu2+, Ag+, and Hg2+ complexes  in the DMF solution were scanned in the range of 190–810 nm at room temperature. The values of the λmax and magnetic moments (μeff) are recorded in 2+, Ag+, and Hg2+ complexes, although Ag compounds were known mostly as two-coordinated, but the square planar silver(I) complexes increasingly observed stereochemistry for silver(I), as was found in our synthesized complex  is a colorimetric examination for measuring the cell metabolic activity and proliferation . In the 2+, Cu2+, Ag+ and Hg2+) prepared complexes were studied. The outcomes revealed the following:The characterization and biological activity of four new  is square pyramidal.Depending on the HOMO-LUMO energy gap, the DFT analysis generated that they were more stable complexes than the parent ligand.Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and Streptococcus mutans with inhibition zones of 65.95 ± 0.5, 57.36 ± 0.6, 69.21 ± 0.6, and 72.34 ± 0.5, respectively, which are higher than the compared reference antibiotics gentamicin (for Gram-negative) and ampicillin (for Gram-positive). Moreover, the ligand and its complexes (1S–4S) exhibit substantial anticancer effects.Ni complexes are the most potent antimicrobial agent against different bacterial strains of 50 values were ordered as follows: vinblastine (4.58) < ligand (20.45) < Ni(II) complex (41.2) < Hg(II) complex (48.5) < Cu(II) complex (182.61) < Ag(I) complex (293.95) µM. This highlights that it might be useful in cancer treatment.The ligand and its complexes are effective against the HepG2 cell line. The ICMolecular docking explored the suggested interactions with the active amino acids of the ribosyltransferase and EGFR tyrosine kinase enzymes.