Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control′s response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and lethal disease associated with aging . Its patHypoxia is a stress condition that influences cell fate by modifying numerous circuits. In this context, especially in the “fibroblast foci”, the main histopathological characteristic of these patients are the mechanisms of hypoxia adaptation that result in profibrotic feedback signaling, which could perpetuate a fibrotic state . For exaTranscriptomic analysis allows us to define the stimulus and landscape of altered circuits . TherefoThe bioethics committees of Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas (INER) approved this protocol . The donation of samples (biopsy) from patients and donors was performed following the relevant guidelines and regulations with the informed consent of all participants, as previously described [2.Cell culture was performed using Ham’s F-12 medium  supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin, 2.5 mg/mL of amphotericin B, and 10% FBS (Gibco Cat No. 11550356), at 37 °C, in an atmosphere of 95% air and 5% COPrimary or commercial cell lines from patients with IPF  and controls  were used; cell demographics are shown in 2, 5% CO2, and balanced with N2. An oxygen analyzer (Teledyne Electronic Technologies 60T) with an oxygen sensor  was used to monitor oxygen concentration.For the experiments under hypoxic conditions, the fibroblasts were cultured in a 60 mm culture dish and subsequently transferred and maintained in a modular incubation chamber , at 37 °C, for 48 h, in a humidified atmosphere with the following mixture of hypoxic gases: 1% O5 cells were used; once washed twice with Phosphate Buffered Saline (PBS) in Petri plates of 60 mm, 600 µL of RLT buffer was added per plate. They were detached with the help of a police rubber, then were transferred to a tube, and were homogenized for at least 45 s at maximum speed; afterwards, 70% ethanol was added in a 1:1 ratio and homogenized well; 700 µL of this mixture was transferred to an RNeasy Mini spin column with a 2 mL collection tube and centrifuged at 9000 rpm/30 s, discarding what was obtained in the tube. Subsequently, 700 µL of RW1 buffer was added to the RNeasy Mini spin column, and it was centrifuged at 9000 rpm/30 s. Again, what was collected was discarded. Later, 500 µL of Buffer RPE was added to the RNeasy spin column, and it was centrifuged at 9000 rpm/30 s. This collection tube was discarded together with what was obtained; a new tube was placed for RNA storage, and 40 µL of RNase-free water was added directly to the spin column membrane and centrifuged at 900 rpm/1 min to elute RNA.Total RNA was isolated with RNeasy Mini Kit  according to the manufacturer’s protocol. Approximately 8 × 10Extracted RNA was quantified spectrophotometrically with NanoDrop ND-1000 . RNA quality was assessed with an Agilent 2100 Bioanalyzer .® WT PLUS Reagent Kit  was used for cDNA preparation and biotin labeling. cRNA was purified using an Affymetrix magnetic bead protocol. The Affymetrix GeneChip Hybridization, Wash, and Stain kits were used for array processing. Arrays were incubated for 16 h in an Affymetrix GeneChip 645 hybridization oven at 45 °C with rotation at 60 rpm. The chips were subsequently scanned with an Affymetrix GeneChip Scanner 3000. Raw data were analyzed using Affymetrix Expression Console and Transcriptome Analysis Console software prior to downstream analysis.Isolated total RNA was amplified, labeled, and hybridized using the Clariom D platform, which detects >540,000 transcripts, following the manufacturer’s instructions . Briefly, the Affymetrix GeneChiphttps://figshare.com/projects/Hipoxia/141623. The microarray data were analyzed using R software version 4.1.0 [Microarray data can be downloaded from the on 4.1.0 , and Bioon 4.1.0 . Qualityon 4.1.0  to minimp-values , and logFC > 1 or logFC < −1.To identify significant differences between gene expression in each condition, we selected the contrasts specified in http://hurlab.med.und.edu:3838/VennDetail/) [Ggplot2 version 3.3.5  was usedDetail/) , for PCAhttps://digitalinsights.qiagen.com/IPA) [The pathways and networks were generated using the differentially expressed genes using QIAGEN Ingenuity Pathway Analysis, version 2000–2022 (com/IPA) . An enricom/IPA) ,25,26.2. For this, three lines of fibroblasts from healthy lungs were used as controls , and three primary lines of lung fibroblasts from patients with IPF  were used to address the heterogeneity of cell lines and the variability in the hypoxic stimuli. Subsequently, the samples were analyzed with the Clariom D platform  and, second, by determining the specific response for each cell line. It is essential to mention that when the dispersion was analyzed in the PCAs, control or fibrotic (C or F) cell lines were clustered together in their respective condition group . Cell liAnalysis by condition revealed a strong transcriptional response to hypoxia in control fibroblasts, which showed 1006 differentially expressed genes (DEGs) A,C. In sNetwork analysis using Ingenuity Pathway Software (IPA) shows a transcriptomic landscape in normal fibroblasts during hypoxia revealing metabolic changes regulated by HIF-1α A,B. ThesTo further evaluate possible directional consequences and inferred upstream regulator activity in HIF-1α signaling in hypoxia in both control and fibrotic fibroblasts, we used the IPA tool Molecular Predicted Map on our data set, where it is also shown that the controls have greater complexity in the signaling pathways .The Venn diagram of The number of unique DEGs that are not shared with any other cell lines is heterogeneous in number; the control lines C1, C2, and C3 have 1412, 209 and 540 genes, respectively A, while The pathways in the group of fibroblasts from controls are related to the epithelium development, differentiation, and pathways involved with several metabolites C. Due toA third Venn diagram was constructed to identify the transcriptional signature with dysregulated genes shared between the controls and IPF A. Of theFive genes were identified to be down-regulated that have not been studied in IPF, which are Cyclin G2 (CCNG2), Potassium Channel Tetramerization Domain Containing 16 (KCTD16), Basic helix-loop-helix family member e41 (BHLHE41), Syntaxin binding protein 6 (STXBP6), and Serpin family B member 7 (SERPINB7) .Nine genes were found up-regulated, four genes have been previously related to IPF: Endothelial PAS domain protein 1 (EPAS1), Transferrin receptor (TFRC), Endothelin receptor type A (EDNRA) and Periostin (POSTN). The other five genes have not been associated with IPF: the Alcohol dehydrogenase 1B (ADH1B), Protocadherin 18 (PCDH18), Homeobox A5 (HOXA5), Solute carrier family 14-member 1 (SLC14A1), and Ubiquitin specific peptidase 18 (USP18) .Hypoxia is active in the lungs and fibroblasts of IPF patients. Several studies support the idea that hypoxia through the hypoxia-inducible transcription factors (HIF-1α and 2α) is a determining factor in the development and progression of fibrosis, participating in fibroblast activation and differentiation ,8,40,41.2 incubator, where oxygen concentration differ from concentration in the microenvironment typical of the fibroblast focus. Therefore, this work also provides information on the possible dysregulated pathways in the fibroblast foci of patients with IPF.Most in vitro studies with lung fibroblasts from patients with IPF are performed under conditions other than the fibrotic microenvironment in a COThis exploratory and descriptive study was carried out to generate hypotheses about the possible signaling pathways involved in the hypoxia adaptation and the altered phenotype of IPF derived fibroblasts.Therefore, we performed a gene expression analysis with a robust platform (Clariom D), where fibroblasts were analyzed under a hypoxia environment. This study shows that hypoxia induces essential changes in the expression of genes in controls related to metabolism, stress response, and epithelial differentiation, a modest response related to angiogenesis and regeneration in the case of IPF. Exclusive transcriptomic signature in IPF fibroblasts suggests a particular adaptation where EPAS1 (HIF-2α) plays a leading role.As expected, the pathways shared between control fibroblasts and IPFs are related to hypoxia signaling. However, the intensity and complexity of the transcriptomic changes vary between the condition and the cell lines. On the whole, in the controls, there are more genes involved in pathways that are essential for cell maintenance in conditions of limited oxygenation, such as autophagy and metabolism. In fibroblasts derived from patients with IPF, a limited activation is observed, and similar results were observed with other stressors such as starvation and apoptosis inducers ,43,44.HIF-1α and 2α are known to have common target genes, yet it has been suggested that each isoform may exclusively regulate some genes ,46,47,48As was foreseeable, the extracellular matrix-related genes are upregulated in hypoxia in both controls and IPF, so they are not exclusive to the specific transcriptional signature of IPF in hypoxia. However, the possibility remains that its increase is grater in IPF.In the transcriptional signature that was observed in the genes exclusive to IPF, unexpected findings occurred, since no previous connection to hypoxia or IPF had been made such as the circadian rhythm and the differentiation of Th17 cells. In the case of the circadian rhythm pathway, it has been reported that oxygen through the activation of HIF1α, is a reset signal for circadian clocks .Among the 19 DEGs found in the transcriptional signature, some genes such as EPAS1, POSTN, HOXA5, and HHIP are related to regeneration and development. We recently proposed that hypoxia signaling pathways are necessary for the context of lung regeneration. In addition, recapitulation of these pathways has been reported in pulmonary fibrosis . HoweverPathways in IPF fibroblasts that are not shared with healthy cells are pathways that could help us begin to guide potential therapeutic targets. In the case of fibroblasts from patients with IPF, high levels of EPAS (HIF-2α) suggest that it plays an essential role in this chronic adaptation to hypoxia.


Intracellular accumulation of abnormal proteins with conformational changes is the defining neuropathological feature of neurodegenerative diseases. The pathogenic proteins that accumulate in patients' brains adopt an amyloid-like fibrous structure and exhibit various ultrastructural features. The biochemical analysis of pathogenic proteins in sarkosyl-insoluble fractions extracted from patients’ brains also shows disease-specific features. Intriguingly, these ultrastructural and biochemical features are common within the same disease group. These differences among the pathogenic proteins extracted from patients’ brains have important implications for definitive diagnosis of the disease, and also suggest the existence of pathogenic protein strains that contribute to the heterogeneity of pathogenesis in neurodegenerative diseases. Recent experimental evidence has shown that prion-like propagation of these pathogenic proteins from host cells to recipient cells underlies the onset and progression of neurodegenerative diseases. The reproduction of the pathological features that characterize each disease in cellular and animal models of prion-like propagation also implies that the structural differences in the pathogenic proteins are inherited in a prion-like manner. In this review, we summarize the ultrastructural and biochemical features of pathogenic proteins extracted from the brains of patients with neurodegenerative diseases that accumulate abnormal forms of tau, α-synuclein, and TDP-43, and we discuss how these disease-specific properties are maintained in the brain, based on recent experimental insights. The pathological hallmark of neurodegenerative diseases is the accumulation of misfolded proteins in neurons and/or glial cells. The accumulation of amyloid-β (Aβ) and tau in the brains of patients with Alzheimer's disease (AD) was reported in the 1980s, and the group of diseases characterized by abnormal tau inclusions is referred to as tauopathies , 315. InC), whereas when converted to the abnormal form (PrPSc), it forms amyloids rich in β-sheet structures. . 286]. TLike tau, insoluble α-syn extracted from the brain of patients with synucleinopathies exhibits prion-like properties and causes seed-dependent α-syn aggregation in vitro and in vivo. Heterogeneity of the prion-like seeding activity has also been reported, depending on the α-syn strain , 314. SiTARDBP gene encoding TDP-43 are associated with the onset of ALS and FTLD-TDP [C9ORF72 gene also cause TDP-43 proteinopathy, suggesting that the interaction of these RNA-binding proteins with TDP-43 and disruption of RNA metabolic homeostasis are involved in the intracytoplasmic accumulation of TDP-43 [TDP-43 is an RNA-binding nuclear protein localized mainly in the nucleus . It consFTLD-TDP . More thFTLD-TDP , 277. Muf TDP-43 , 243. Ref TDP-43 , 81. Undf TDP-43 , 95, 187Immuno-electron microscopic studies of brain sections from cases of ALS and FTLD-with ubiquitin-positive inclusions have shown the presence of TDP-43 and ubiquitin-positive bundles of straight filaments 10–20 nm in diameter in neurons , 188. AmE. coli as substrates [GRN or C9ORF72 gene mutations into Tg mice expressing human TDP-43 in the cytoplasm reproduced TDP-43 pathology associated with the inoculated samples in a time-dependent manner [Patient-derived insoluble TDP-43 exhibits prion-like properties in vitro and in vivo. An RT-QuIC study found that abnormal TDP-43 in brain homogenates and CSF derived from ALS and FTLD cases were amplified using full-length TDP-43 and truncated TDP-43 (residues 263–414) purified from bstrates . Abnormabstrates . Furtherbstrates . Insolubbstrates , 176. Inbstrates . In vivot manner . Inoculat manner .As we have described, abnormal proteins that accumulate in the brains of patients exhibit ultrastructural and biochemical properties that are characteristic of each disease. It is worth emphasizing that these properties are common in the same disease group, and even in different brain regions of one individual. These results suggest that pathogenic tau, α-syn and TDP-43 spread throughout the brain, retaining their disease-specific structural and biochemical properties.To further explore this possibility, we examined whether disease-specific tau aggregation and filament formation were reproduced in cultured cells . InsolubThe requirement of amino acid sequence identity between template and substrate for template-dependent amplification is also clearly demonstrated by the in vitro and in vivo observation of low seeding and propagation efficiency caused by species barriers , 251. SeThe heterogeneity of abnormal proteins accumulated in patients’ brains is a key factor in the onset and progression of neurodegenerative diseases. Structural and biochemical analyses of post-mortem brains have revealed structural polymorphisms (strains) and prion-like properties of pathogenic proteins. Notably, recent cryo-EM studies of tau and α-syn filaments extracted from patients’ brains have provided direct evidence of strain formation in the brain Table . Detaile


The aim of the HEARTS in the Americas initiative is to promote the adoption of global best practices in the prevention and control of cardiovascular diseases, and improve the control of hypertension. HEARTS is being implemented in 21 countries and a diverse set of actions and measures are in progress to improve exclusive access in primary health care facilities to automated blood pressure measuring devices that have been validated for accuracy. The purpose of this manuscript is to illustrate these efforts, mainly in the regulatory and public procurement arena, and to present information on common challenges and solutions identified. Examples from six countries confirm the need for not only a robust regulatory framework to increase availability of validated automated blood pressure measuring devices but also a comprehensive strategic approach that involves relevant stakeholders, includes a multi-pronged approach and is associated with a national program to prevent and control non communicable diseases. High blood pressure (BP) is a leading risk factor for cardiovascular disease, which is a major health burden on the Region of the Americas . HEARTS In work by PAHO and countries implementing HEARTS, regulatory deficiencies have been identified that allow automated BPMDs to be marketed without evidence of accuracy validation , which iSince 2017 the number of countries implementing HEARTS in the Americas has grown, and there are currently 21 countries enrolled in the initiative at different stages of implementation and level of expansion. The efforts to improve access to validated automated BPMDs are different for each country, in line with its priorities, health system maturity including regulatory, economic, and political conditions. Nevertheless, there are common themes between countries for successes, challenges and the steps taken improve access to validated automated BPMDs. The purpose of this manuscript is to provide examples from six selected countries that are trying to address the problem of non-validated automated BPMDs. The information contained in this manuscript may be useful for other countries that are considering changes to regulatory and procurement processes with the goal of achieving exclusive use of automated BPMDs clinically validated for accuracy.In order to assist the transition towards exclusive use of validated automated BPMDs, and in line with the WHO recommendations , the PanAny modifications aiming to improve regulations or procurement mechanisms related to automated BPMDs need to be coordinated, progressive and sustainable and therefore must be integrated into a national health strategy and cardiovascular health programs. Policies should also be flexible enough to account for technological innovations and advancement of research methods.Box 1 Elements of HEARTS in the Americas strategy to ensure exclusive use of validated automated blood pressure measuring devices (BPMDs) in primary health care facilitiesUnderstanding the regulatory context through mapping of regulatory bodies and processes.Creating awareness by bringing the concept and know-how of accuracy validation into the public health agenda.Training on protocols for accuracy validation of automated BPMDs to enable countries to perform studies and educate on the difference between accuracy validation and other mechanisms of quality assurance.Providing information on which automated BPMDs are validated and available in the Region of the Americas for acquisition by the public sector.Developing technical specifications for procurement of automated BPMDs to be used as criteria for procurement and for regulations.Guidance and technical support in the development, implementation and monitoring of regulations.Promotion of a process that counts on the ample participation of key stakeholders and that is implemented gradually.Different approaches to improve access to validated automated BPMDs have been taken by governments of each country, based on their priorities, regulatory systems, political and economic characteristics. Examples of key actions and challenges specific to Brazil, Chile, Cuba, Dominican Republic, Ecuador and Saint Lucia were provided by representatives of governments  of selected HEARTS countries. The narratives provided were checked against other information previously obtained and online records for consistency, condensed and edited for clarity and validated by the coauthor representatives . An overview of the common strengths, challenges, key actions and lessons are summarized in Table Accuracy validation based on ISO 81060-2: 2018 or other validation protocols is currently required for the marketing of automated BPMDs in Brazil. Manufacturers (or importers) must submit a clinical investigation report to the National Institute of Metrology (INMETRO), which analyzes the report content to confirm that all points required by the protocols are met. At INMETRO, investigation reports are reviewed by metrology researchers, generally with a background in engineering and experience in metrology, focusing on healthcare.The accuracy validation study results are provided directly by the manufacturer. However, the results are not required to be included on registries of validated automated BPMDs or published in peer-reviewed scientific journals. This suggests a lack of independence of the manufacturer from the accuracy validation data, which is not recommended as best practice , 16. ForThe implementation of the HEARTS Initiative in 2017, in which Chile was part of the first cohort, has reinforced the need to improve the quality of BP measurement. Since 2012, the purchase of validated automated BPMDs was recommended by the Ministry of Health (MoH). This recommendation was stressed in 2018 by HEARTS implementation with the development of the BP measurement protocol, videos of correct BP measurement and educational materials for patients, all emphazing that automated BPMDs must be validated for accuracy. The MoH’s National Supply Center reported that between 2018 and 2020, automated BPMD purchases increased from 15% to 75% of the total number of devices. Likewise, the proportion of validated automated BPMDs acquired for use in the public sector increased from 34% in 2018 to 64% in 2020. It is important to note that there are other purchasing mechanisms, such as those defined by the different Health Services , which are autonomous to make their own decisions.In 2021, a BP measurement protocol was published on the MoH website stressing the recommendation of only using validated automated BPMDs . In addiChile still has challenges from the regulatory perspective because the measurement protocol and the recommendations for acquisition of automated BPMDs aforementioned are not normative yet and thus are not enforceable. To address this challenge, a group including representatives of government bodies responsible for the purchase and surveillance of medical devices across Chile is currently working on a regulation proposal that includes impact assessment and identification of the automated BPMDs available in all health care facilities across the country.Cuba cannot access validated automated BPMDs available in the global market due to their high cost and other economic and trade restrictions. Therefore, the validated automated BPMDs available as of December 2021 were obtained mainly through international technical cooperation projects. However, a Cuban medical equipment manufacturer has developed automated BPMDs for 24-hour ambulatory monitoring and, as of 2016, an automated BPMD for home and office use. The locally produced automated BPMDs are currently used at all levels of care. In 2018, due to the HEARTS Initiative emphasis on replacing aneroid devices, domestic automated BPMDs were introduced and rapidly expanded to provide at least one device for approximately 70% of the 11,000 PHC teams in the 489 health centers across the country. However, those devices are not yet clinically validated using the ISO 81060-2:2018 standard or any other validation protocol.Catalyzed by the HEARTS in the Americas, in 2019, training on accuracy validation studies was provided by experts from the University of Alberta, Canada. The purpose of the training was to develop the technical expertise and knowledge to conduct validation studies according to the highest international standards and assess the accuracy of the locally produced automated BPMDs. Subsequently, the National Hypertension Commission, an independent technical body, approved the formation of a technical group to conduct an accuracy validation study under the academic coordination of the Institute of Cardiology and Cardiovascular Surgery, with the participation of the State Center for the Control of Medicines, Equipment and Devices, a WHO collaborating center, to verify the accuracy of locally produced automated BPMDs. There was no industry participation in any step of the process.In parallel to the accuracy validation effort, the conditions will be created to modify the regulatory framework to incorporate ISO 81060-2:2018 into Cuban Standards to ensure exclusive use of validated automated BPMDs before 2025. Finally, the availability of automated BPMDs must be improved, including expanding the production of the current model and the development and commercialization of new ones, with more capabilities, such as data transmission features.A challenge in the Dominican Republic is the precariousness of the electrical power supply in many remote health centers and the absence of uninterrupted availability of batteries to operate automated BPMDs. For these reasons, the HEARTS national managing team chose to temporarily maintain the use of aneroid BPMDs in some facilities but also to provide additional training for health personnel focused on the correct use of automated BPMDs. Simultaneously, an exhaustive search was conducted to identify automated or semi-automated BPMDs that would not need batteries to operate. The search led to choosing a validated semi-automatic BPMD with rechargeable batteries that is accurate in pregnancy and meets the WHO criteria for use in a low-resource setting . This deSince this was the first time a digital device would be used in the country, a 15-day study was conducted with a small sample of health personnel to explore how they would respond to this change. This study, which is yet to be published, was conducted by taking measurements alternatively with aneroid devices and the semi-automated BPMDs on the same patient. There was some resistance to change by the medical profession. To overcome this resistance progressively, health care personnel are being trained through local HEARTS workshops and the HEARTS BP measurement course, to educate them on the difficulty of accurately diagnosing hypertension with an aneroid sphygmomanometer.In 2018, 94 validated automated BPMDs were acquired to conduct the Ecuador non-communicable diseases risk factors survey and PHC facilities that had started implementing HEARTS. Unfortunately, budget cuts within the Ecuadorean MoH have since hindered the purchase of new validated automated BPMDs. However, the initial devices were followed by the acquisition of an additional 468 validated automated BPMDs, including 300 devices through liaison with the HEARTS coordinating team and international cooperation agencies. As of December 2021, 352 health centers were implementing HEARTS, which corresponds to 18% of the country’s 1940 health centers. Approximately 85% of these 352 centers have validated automated BPMDs, which means 15% of the total in the country.In addition, the HEARTS in the Americas pillar on education and training has found great receptivity demonstrated by the high number of health professionals taking the HEARTS virtual course on BP measurement . The nurTo ensure exclusive public procurement of validated automated BPMDs for HEARTS implementing centers, technical specifications that include international accuracy validation standards have been developed by the MoH with the support of. In addition, the process to make the standards enforceable for the acquisition of BPMDs for use in any public facility, especially those in PHCs, has been initiated within the MoH and is expected to be approved in the first trimester of 2022.A challenge has been the limited availability of validated automated BPMDs in the national market. This was identified when procuring BPMDs for use in HEARTS implementing centers in 2021, by analysis of the National Agency for Regulation, Control and Sanitary Surveillance (ARCSA) database of BPMDs with pre-market approval for sale. Only one had presented proof of passing accuracy validation testing from the five models registered for sale. An additional challenge is the lack of knowledge of the advantages of using automated BPMDs and preventive maintenance to extend their useful life. Provision of information and advocacy with health care personnel and managers are being conducted to address these issues. However, a key step will be modifying regulations to include accuracy validation as a requirement for registration at ARCSA, which is underway.In Saint Lucia, registration of automated BPMDs for sale in the country is dependent upon approval by the Saint Lucia Bureau of Standards (SLBS), which has responsibility for enforcement of the Metrology Act. The Act is a law which gives the SLBS responsibility for approval and verification of measuring instruments used in the field of public health, including automated BPMDs. However, achieving the goal of enacting legislation that would ensure the exclusive sale of accuracy validated automated BPMDs in the country involves other government sectors, and, to that end, discussions between the stakeholders, such as MoH, Ministry of Commerce, Attorney General’s Chambers, and Customs, have been underway.Meanwhile, in May 2021, accuracy validation became a prerequisite for verification of accuracy, performance, and calibration by the SLBS, as per OIML R149 requirements. A campaign to promote the use of validated BPMDs has been carried out with the use of social media, and two webinar presentations were given by the SLBS to the medical sector and importers recommending that only validated automated BPMDs approved for sale in the country and tested for accuracy against established protocols be used. One large pharmacy chain in Saint Lucia imported a stock of validated automated BPMDs for sale to the public as a result of an SLBS presentation to the medical sector and it is anticipated that competitive forces will play a role in ensuring that other suppliers follow suit. The public service announcement from the SLBS on the verification of automated BPMDs, which first aired in 2017, and is presently on the SLBS’ YouTube channel will be edited to include specific information on validated automated BPMDs.Among the challenges are online purchases of non-validated automated BPMDs at distributors and retail outside Saint Lucia, pressure from suppliers to sell the remaining stock of non-validated automated BPMDs and the variability of information available on online registries.The six countries that were the focus of this manuscript have shown the political will to reach the exclusive availability of validated automated BPMDs. Indeed, most of the facilities implementing HEARTS began to use accuracy-validated automated BPMDs, and this represented an important stimulus to advance this strategic work. The general achievements by these six countries showed in concrete actions are twofold: (a) sectors of governments have been investing politically and administratively in modifications to regulations that do not require a substantive and complex legislative process; and (b) in parallel, improved procurement toward exclusive access of validated automated BPMDs has been at least partially achieved. The improvement of procurement mechanisms has focused on defining technical specifications that include accuracy validation. In addition, lack of access to validated automated BPMDs has been counteracted by local production of devices and cooperative procurement approaches. The lessons shared in Table There are many ongoing challenges confronted by countries implementing HEARTS. The common challenges are related to changing regulations and/or legislation, budgetary limitations and resistance to uptake of automatic BPMDs by health professionals, particularly physicians Table . Steps tThe key next step for countries is to develop, implement and enforce regulations that guarantee that only those automated BPMDs that have passed an accuracy validation study are approved for sale in the country. This step will require the participation of different actors. It may need to start with the education and advocacy actions, providing information on the benefits of using validated automated BPMDs to policymakers, procurement officers, health care providers and managers of health services. In addition, advocacy involving medical and professional societies, patients (or consumers) groups and civil society may create pressure to improve the quality of care. These processes, which are underway to different degrees in each country, may require understanding the audience, timing, and engaging with real-world policy-making .Another next step that will improve access to and knowledge of validated automated BPMDs is the development of country-specific lists or a regional list of validated automated BPMDs relevant to the Region of the Americas. A key feature of this list, in addition to the standard specifications of devices, should be information about the power source of the validated automated BPMDs, because access to reliable power varies greatly across the Americas. There has been a substantial increase of awareness of the importance of using accuracy validated automated BPMDs in countries implementing HEARTS. However, there are still several gaps in knowledge of technical aspects of automated BPMDs, which need to be summarized and disseminated to procurement officers.The challenges that the countries face to reach the goal of exclusive use of validated BPMDs in PHC are enormous and multifactorial. This confirms the need for a robust regulatory framework and comprehensive strategic approach to exclusive use of clinically validated automated BPMDs at regional level. HEARTS in the Americas has adopted a strategy to overcome these challenges over time, concomitantly conducting parallel efforts in several areas. The successful implementation of the initiative depends on a coordinated effort across government and other stakeholders that will contribute to better diagnosis and management of hypertension in the Americas.Blood pressure measuring devices that have been clinically validated have shown to be more accurate than those that have not.Most blood pressure measuring devices found in the global market have not been clinically validated.The regulatory framework pertinent to the marketing of blood pressure measuring devices in the Region of the Americas is weak and fragmented. There is a need to implement regulations and procurement mechanisms to ensure exclusive use of validated measuring devices.There are multiple strategies to reach the goal of exclusive use of validated automated blood pressure measuring devices, and these should be adopted concomitantly to the extent of their feasibility.The diversity of challenges faced by countries are related to their political and economic landscape, health systems and regulatory maturity, and factors such as geography and population size.The different approaches to these challenges describe in the paper could be used as lessons by other countries in their efforts to reach the goal of exclusive use of validated automated blood pressure measuring devices in primary health care.


Past work has focused on the thermal properties of microscale/nanoscale suspended/supported graphene. However, for the thermal design of graphene-based devices, the thermal properties of giant-scale (~mm) graphene, which reflects the effect of grains, must also be investigated and are critical. In this work, the thermal conductivity variation with temperature of giant-scale chemical vapor decomposition (CVD) graphene supported by poly(methyl methacrylate) (PMMA) is characterized using the differential transient electrothermal technique (diff-TET). Compared to the commonly used optothermal Raman technique, diff-TET employs joule heating as the heating source, a situation under which the temperature difference between optical phonons and acoustic phonons is eased. The thermal conductivity of single-layer graphene (SLG) supported by PMMA was measured as 743 ± 167 W/(m·K) and 287 ± 63 W/(m·K) at 296 K and 125 K, respectively. As temperature decreased from 296 K to 275 K, the thermal conductivity of graphene was decreased by 36.5%, which can be partly explained by compressive strain buildup in graphene due to the thermal expansion mismatch. A massive amount of work has focused on the experimental and theoretical calculation of the k of suspended and supported graphene, and great progress has been achieved [k of graphene has been deeply studied. Last but not least, since temperature affects the phonon propagation, the k of graphene shows strong temperature dependence. It has been reported that the k of supported graphene increases with increasing temperature, with the peak k appearing around 300 K [The discovery of graphene created explosive growth in research on the various properties of graphene ,2,3,4. Dk) , ultrahik) , and remk) , graphenk) ,9, photok) , batterik) , and so k) . With thachieved ,15,16,17achieved ,19,20, sachieved ,22, and achieved ,24,25 onnd 300 K . When thnd 300 K .k depends on the absorbed laser power and the temperature coefficient of the Raman shift of the G peak [k of graphene will be overestimated. Wang et al. first detected the lumped temperature difference between optical and acoustic phonons by nanosecond energy transport state-resolved Raman spectroscopy [k of supported graphene. The micro-bridge method uses joule heating as the heating source. In the experiment, the graphene supported by SiO2 acts as the thermal bridge between the heating part and the temperature-sensing part [Thermal property measurement is critical to the application of graphene in thermal management. Since graphene is extremely thin, the commonly used thermal conductivity measurement techniques of graphene are optothermal Raman spectroscopy ,27,28 ane G peak . Li et ae G peak ,30. It ie G peak . Under ttroscopy . Seol eting part . In prining part .k of suspended graphene is size/geometry dependent. The k of graphene has a ~log L (L: sample length) dependence which was confirmed by experiments (up to 9 μm) [k of supported graphene is less sensitive to the length size due to substrate coupling [k of macroscale supported graphene is critical, since the edge-phonon scattering usually leads to a great reduction in k. Furthermore, graphene has important applications in the field of flexible electronics, which calls for the thermal properties of graphene supported by flexible materials. To date, seldom work has been conducted on the temperature dependence of the thermal conductivity of millimeter-scale graphene on soft substrates, yet such knowledge is essential to graphene-based device design and optimization.So far, most thermal conductivity measurements of graphene focus on microscale samples. It is reported that the to 9 μm)  and theoto 9 μm) . Althougcoupling ,35, the k of PMMA is much lower than that of graphene. Choosing extremely thin (~457 nm) PMMA as the substrate can ensure the contribution of graphene to the overall thermal conductivity is comparable to that of PMMA. Thus, it is feasible to measure the k of graphene with high accuracy. The k of supported SLG is reported to be 743 ± 167 W/(m·K) at 296 K, which is 23.8% higher than that of micro-scale SiO2-supported graphene measured using the micro-bridge method [k of supported SLG is reduced to 287 ± 63 W/(m·K) when the temperature is 125 K, partly due to the strain-induced rips in the sample.In this work, the temperature dependence of the thermal conductivity of millimeter-scale SLG supported by poly(methyl methacrylate) (PMMA) is characterized by a differential transient thermoelectrical technique (diff-TET) . The dife method . The k o2 flow rate of 500 sccm. Then, the H2 flow rate and chamber pressure were adjusted to 70 sccm and 108 Torr, respectively. By introducing 0.15 sccm CH4 into the chamber, the graphene starts to grow. The copper was etched off after PMMA was coated on the graphene. Finally, graphene supported by PMMA was transferred to a filter substrate. The layer number of graphene was provided in the product technical data.SLG supported by PMMA characterized in this work was obtained from Jiangsu Xianfeng nanoscale materials company. The samples used in this experiment were fabricated using the chemical vapor deposition (CVD) method. The graphene is grown on a copper (Cu) in a controlled chamber pressure CVD system. At first, a clean Cu foil was annealed at 1077 °C with a Hk of these samples will be measured by diff-TET without any further processing. The thickness of PMMA (pδ) is necessary to determine the k of graphene when subtracting the effect of PMMA. Atomic force microscopy (AFM) was used to determine the thickness of PMMA. The supported graphene was transferred to a silicon substrate; then, the thickness of PMMA was determined with AFM. The as-received graphene was cut into the desired size by scissors. Then, the experimental sample was suspended between two electrodes, as shown in −1) can be observed in the Raman spectra, indicating that the supported graphene has rare D band-related defects. Peaks at 1588.3 cm−1 (G band) and 2681.5 cm−1 (2D band) can be observed. We used a ratio (IG/I2D) of the intensity of the G band to the intensity of 2D band to evaluate the layer number of the as-received sample [Even though the layer number of the as-received sample was given in the technical data sheet, the layer number of the as-received graphene on PMMA was verified with confocal Raman spectroscopy . In total, 30 random spots were tested. In the Raman spectroscopy test, a 532 nm laser with ~0.45 mW was focused on the graphene under 50× objective. The integration time was 10 s. Spectra of the as-received sample are shown in d sample . It was V0 and V1 are the initial voltage and steady-state voltage over the sample, respectively. The environmental temperature was controlled by the temperature controller which ranges from 7 K to 300 K. Since most of the samples were broken at 125 K with a sudden jump resistance to a very high value, we did the experiments from room temperature (RT) down to 125 K in 25 K steps. During the joule heating, the thermal transport in the sample can be regarded as one-dimensional, thus the energy governing equation can be expressed as:ρ, pc, and k are the density, specific heat capacity of the sample, respectively. A and L are the cross-sectional area and length of the sample, respectively. I and sR are the fed-in current and the sample resistance, respectively. Since the two electrodes are much larger than the sample, the temperature of the two electrodes can be regarded as environmental temperature. Thus, the boundary conditions can be depicted as: T = T0 (T0: room temperature). The theoretical normalized temperature rise is defined as: The thermal diffusivity of graphene supported by PMMA was characterized by diff-TET . As showeffα which gives the best fit of the experiment data was taken as the sample’s effective thermal diffusivity. A Matlab program created based on Equation (2) was used to fit the normalized temperature rise obtained by the experiments. The trial value of effα) includes the radiation effect (radα) which can be expressed as:ε, Bk, and δ are emissivity, the Stefan–Boltzmann constant, and thickness of the sample, respectively. The emissivity of the sample is taken from [realα), the effective thermal conductivity (effk) can be obtained as effk = realα·(pρc)p. The volumetric heat capacity of PMMA was used for the whole sample with high accuracy due to the extremely low mass proportion of graphene. In part 2, the Raman spectroscopy study of graphene showed that the graphene is distributed on the PMMA uniformly and all the graphene in samples is found to be a single layer. The relationship between effective thermal conductivity and thermal conductivity of PMMA and graphene can be depicted as:The effective thermal diffusivity  between graphene and PMMA is neglectable in comparison to the thermal resistance (~106 K/W) of PMMA. More details can be found in [Here, ferences ,39. The ectively ,39. The ectively . Additiofound in .R* = R/R0, R0: resistance at RT) variation against the temperatures of S1, S2, S3, and S4 is presented in eρ) of graphene is linearly proportional to T, as temperature is higher than the Bloch–Gruneisen temperature (BGΘ) of graphene and is proportional to T4 in the opposite case (BGT < Θ) [R* of the four samples decreases slightly as temperature decreases, then increases as temperature is decreased from RT to 125 K. The turning point is in the range of 250–275 K. The R* of S2 and S4 is increased by 35% and 23%, while the R* of S1 and S3 is decreased by 0.012% and 0.0075% as temperature changes from RT to 125 K. To obtain more details on the resistance of supported graphene, the temperature dependence of R* of S5 was measured every 10–25 K for three rounds. R* trends of S5 are similar to that of S1 and S3. As temperature continues to decrease from 150 K, the R* of S5_r1 and S5_r2 first decreases and then increases with temperature, while the R* of S5_r3 continues to increase. Hinnefeld et al. studied the resistance of graphene supported by PDMS under strain and it was found that the tensile strain in the supported graphene can lead to rips in the graphene. These rips cause the resistance of the supported graphene to increase significantly with increased strain [The normalized resistance (T < ΘBG) . Howeverd strain . As tempγ are the intrinsic TCR of relaxed graphene and a positive constant, respectively. pβ and gβ are the thermal expansion coefficients (β) of PMMA and graphene, respectively. pβ decreases with decreasing temperature and remains positive when the temperature is above 0 K. gβ remains negative when temperature is lower than RT. In the entire temperature range, pβ is relatively large, which helps maintain a positive TCR in supported graphene. As temperature is decreased, the difference between pβ and gβ becomes smaller in comparison to the effect of The temperature coefficient of resistance (TCR) of graphene supported by PMMA is further discussed based on the data of S5_r3. The TCR of S5_r3 is shown in negative ,43. Whenrealα of S1, S2 and S4 decreases first, then the realα of S2 remains constant while the realα of S1 and S4 increases slightly. The change in the realα of S3 is relatively small when compared to the other three samples. The k of SLG supported by PMMA is presented in k of SLG supported by SiO2 [k of supported SLG is 743 ± 167 W/(m·K), which is smaller than that of SiO2-supported SLG (840 W/(m·K)) [k of supported graphene is measured by a comprehensive Raman optothermal method which eliminated the laser absorption uncertainty [k of supported graphene is overestimated. The k in this work is larger than two literature values (~600 W/(m·K) for SiO2-supported SLG [k of our work and above two differences may be attributed to the different interface coupling strength.The real thermal diffusivity without the effect of radiation of four samples is shown in  by SiO2 ,27 and b by SiO2  are showrted SLG  and 560 rted SLG ). The dik of supported SLG first decreases by 36.5%, which is much larger than the k reduction in [k of graphene supported by a flexible substrate. As such, we turn our attention to the effect on the k of suspended graphene. As reported in the literature, compressive strain in suspended graphene suppresses thermal conductivity [k can be reduced by 26% [k reduction when temperature decreases from RT to 275 K. Thus, Raman spectroscopy is used to investigate the G peak under different temperatures. The graphene sample suspended between two electrodes was placed in a cryogenic cell where the temperature can be controlled between 77 and 300 K. A confocal Raman is used to collect the Raman spectra of graphene under different temperatures.As temperature decreases from RT to 275 K, the ction in ,27. As ductivity . This isuctivity ,44. Withd by 26% . We specGω) of SLG on PMMA as a function of temperature. The G peak blueshifts as temperature goes down from RT. The temperature dependent frequency shift of the G peak is attributed to three parts: thermal expansion of the lattice ΔωGΔωGS(/% at RT . Since tulations  are subtgraphene . Howevere strain . For graany rips ,42. At lR–T relationship is due to the TEC mismatch between graphene and PMMA and rips in graphene appearing at lower temperature. Using the diff-TET technique, the temperature dependence of the thermal conductivity of graphene supported by a flexible substrate is obtained. Through monitoring the blueshift of the Raman peak, compressive strain is confirmed to be built up in the graphene. The large reduction in k as temperature decreases from RT to 275 K is partly due to the compressive strain built up in graphene. This work can provide some guidance on the design of graphene-based electrothermal devices.In summary, we first reported the resistance–temperature relationship for supported graphene since this relationship is critical for explaining the behavior of graphene in thermal characterization. The TCR of our sample reduced from a positive value at RT to a negative value at low temperatures, while the free-standing graphene has a negative TCR throughout the entire temperature range. The abnormal


The possibility of integrating thismaterial into two different transducing schemes for gas sensing isproposed since the nanomaterial changes both its electrical resistivityand optical properties when exposed to gases and at room temperature.Particularly, when implemented in a chemoresistive device, the Mo6@Graphene hybrid showed an outstanding sensing performancetoward NO2, revealing a limit of quantification of about10 ppb and excellent response repeatability (0.9% of relative error).While the Mo6@Graphene chemoresistor was almost insensitiveto NH3, the use of an optical transduction scheme (changesin photoluminescence) provided an outstanding detection of NH3 even for a low loading of Mo6. Nevertheless, thephotoluminescence was not affected by the presence of NO2. In addition, the hybrid material revealed high stability of itsgas sensing properties over time and under ambient moisture. Computationalchemistry calculations were performed to better understand these results,and plausible sensing mechanisms were presented accordingly. Theseresults pave the way to develop a new generation of multi-parametersensors in which electronic and optical interrogation techniques canbe implemented simultaneously, advancing toward the realization ofhighly selective and orthogonal gas sensing.This paper reportsfor the first time a gas-sensitivenanohybridbased on octahedral molybdenum iodide clusters supported on grapheneflakes (Mo This is why automated air quality control stations arefew and sparsely distributed in specific areas. However, effectivepollution monitoring can only be achieved through a network of denseranalysis locations (higher granularity) distributed over wider areas.2 In other words, the ubiquitous, unattended, andreal-time detection of harmful gases would protect human health byallowing the taking of pertinent actions in space and time. In thisperspective, the development of gas sensors with the ability to monitorlow concentrations of flammable, combustible, or toxic gases in realtime is crucial. These devices should gather essential propertiessuch as reliability, sensitivity, selectivity, repeatability, accuracy,and long-term stability together with inexpensiveness and low powerconsumption.Air pollution is probably the largestenvironmental health threatworldwide, accounting for 7 million premature deaths every year.3 For decades, metal oxide (MOX) semiconductors have dominated thefield of gas detection because of their high sensitivity, non-toxicity,and availability.4 However, their poorselectivity and power-hungriness (operation temperatures are highabove room temperature) are still preventing their effective implementationin commercial applications for air quality monitoring stations orunattended gas sensing networks. Therefore, the development of newand energy-efficient gas-sensitive nanomaterials is still much needed.Among the different technologies for developinggas sensors, chemoresistivedevices have attracted great research efforts owing to their simpledriving and readout instrumentation, durability, high potential forminiaturization, and industrial scalability.5 Room-temperature-operated gas sensors are energy efficient and helpreduce fabrication costs (as no heating elements are needed), whichmakes them very attractive for being integrated into air quality monitoringnetworks. Despite the noteworthy properties of graphene, such as highsurface area to volume ratios, low noise levels, high carrier densityand mobility, and affordable production costs,6 there are still some issues to be overcome. Graphene, especiallyin its pristine form, shows limited sensitivity and selectivity. Inthis sense, large research efforts have been focused on modifyingor functionalizing carbon-based films for improving their sensingproperties. The most direct functionalization strategies are probablythe modification of graphene via plasma treatments for creating defectsand replacing carbon atoms with others of different nature,7 or the use of wet chemistry processes for thecovalent or non-covalent functionalization of the graphene surface.8 With these approaches, the sensitivity of thegraphene layers is generally improved, but usually the selectivityissue remains only partially addressed, owing to the poor specificityof the atoms and functional groups present on the graphene surface.Another well-known strategy is the functionalization of graphene withorganic compounds such as macrocycles, aptamers, or cavitands.10 This approach significantly improves the selectivity because ofthe high specificity of the organic compounds  toward specific gases. However, the organic nature of thisfunctionalization, which is prone to degradation under operationalconditions, may compromise long-term device stability. Additionally,high specificity may result in limited reversibility of the gas–solidinteractions, which compromises response repeatability. Finally, thelast mainly used strategy is the decoration of graphene with metalor MOX nanoparticles.12 With that, it is possible tosignificantly increase the sensitivity and tune the selectivity tosome extent. However, these MOX nanoparticles usually need moderateor high operating temperatures for activating their sensing properties.Therefore, the achievement of low-power and inexpensive devices canbe jeopardized.In recent years, carbon nanomaterial-based sensors, especiallythose employing graphene, have become a common approach for developingdevices that operate at room temperature.15 These nanostructured semiconductors vary in composition and morphology,which impacts their sensing performance.17 Besides, the fabrication of molybdenum sensors is attractive dueto their abundance and low toxicity, the development of thin filmsand nanoparticles being the most common strategy.18 However, there is significant scope remaining to explorenew molybdenum materials with advanced properties for large-scalefabrication. Particularly, materials composed of architectured nanometer-sizedoctahedral molybdenum clusters (<2 nm) are very attractive forgas sensing purposes.19 These [Mo6Li8La6] cluster unitsare robust entities composed of inner halide ligands (Li) and organic or inorganic apical  ligands (La). Mo6 clusters act as powerful photosensitizers and arealso efficient luminophores that, under photoactivation, either exhibitphosphorescence in the 550–900 nm window or react with oxygento provide singlet molecular oxygen (1O2) directedto cell damage by oxidative stress and to theranostic applications.Until now, the existing literature on analyte sensing using Mo6 cluster-based nanomaterials encompasses optical oxygen sensors,24 optical sensing of antibiotics and nitroaromatic compounds in solution,26 and cluster dependence on environmental parameters, such as humidityor light.28 However, Mo6 clusters have notbeen applied to the detection of air pollutants until now, and theresearch of the materials composed of Mo6 clusters andgraphene and graphene oxide was limited to the photocatalytic field.32As an alternative, molybdenum chalcogenidesand their derivativeshave been extensively explored for gas detection in the last few years.6@Graphene nanohybridcomposed of a crystalline octahedral molybdenum iodide cluster materialsupported on graphene, and the integration of this hybrid onto a transducingplatform, enabling chemoresistive and optical gas sensing. The detectionof two gaseous species such as nitrogen dioxide (NO2) andammonia (NH3) is studied. NO2 is an electron-withdrawingcompound mainly released by automotive emissions and combustion ofconventional fossil fuels, which has become a major environmentalconcern because it contributes to the formation of photochemical smog,acid rain, and particulate matter through chemical reactions in theatmosphere. Ammonia emissions are associated with industrial livestock,fertilizer production processes, and crop agriculture. As a highlytoxic and corrosive agent, they threatenhuman health and the environment.33 Besides reporting the sensing properties ofthe Mo6@Graphene hybrid nanomaterial, its thorough morphologicaland compositional characterization is reported as well. Finally, thegas sensing mechanisms involved in the chemoresistive and opticaldetection are presented and discussed.To the best of our knowledge, the use of hexanuclear molybdenumcluster nanomaterials for the detection of pollutant species in thegas phase remains unexplored. Therefore, this paper encompasses, forthe first time, the preparation of a Mo6Ii8(OH)a4(H2O)a2]·2H2O (Mo6) material and its derived nanohybrid (Mo6@Graphene)were prepared and analyzed using several techniquessuch as Raman spectroscopy, powder X-ray diffraction, field-emissionscanning electron microscopy (FESEM), and high-resolution transmissionelectron microscopy (HR-TEM). The synthesis of Mo6 wasachieved by the hydrolysis of (Bu4N)2[Mo6Ii8(O2CCH3)a6].29 The immobilization of Mo6 on graphene was doneby mixing the respective precursors dispersed in dichloromethane andconfirmed through Raman spectroscopy , resulting in Mo6@Graphene. In this regard, the Ramanshifts below 360 cm–1 are associated with the presenceof molybdenum clusters present in the Mo6 material , while at higher frequencies, thecharacteristic D, G, 2D, and D′ bands of graphene appear at1347, 1588, 2712, and 2938 cm–1, respectively. TheD band is associated with the presence of structural defects, disorderedcarbon atoms in the sp2 configuration, and carbonaceousimpurities, while the G band is related to in-plane vibrations ofsp2 carbon bonds. Thereby, the D/G ratio revealed low crystallinegraphene, and the presence of defects and oxygenated functional groupsgrafted to the graphene is probably supporting the immobilizationof molybdenum clusters. The immobilized inorganic clusters at thegraphene surface are essential in gas sensing performance, enablinghigher interactions with gas compounds. Furthermore, the integrityand crystallinity of the cluster-based materials after the thermaltreatment (60 °C) needed for the preparation of the resultingMo6@Graphene were studied. Figure S1b shows intense bands with Raman shifts at 129, 153, 292, and 357cm–1 associated with the Mo–Mo, Mo–I,and Mo–O vibrations of the cluster.34 These Raman shifts are similar to those reported for similar hexamolybdenumclusters with iodide materials, revealing that the thermal treatmentapplied during the experimental protocol does not damage the clustercrystallinity owing to the lack of differences between both spectra.The suitable cluster synthesis and integrity after the thermal treatmentwere also confirmed through powder X-ray diffraction. Figure S2a depicts the diffractogram of the Mo6 material, which shows a family of planes associated witha preferential orientation of the crystalline material upon depositiononto the glass surface.29 The Mo6 crystallinity was preserved after the thermal process applied duringthe deposition step . Figure S2c shows the diffractogram of the Mo6@Graphene after the deposition process. Considering the lowgraphene crystallinity, it is noteworthy that some intense peaks associatedwith the crystallinity of Mo6 can be noticed in the nanohybrid,evidencing the presence of supported cluster crystals. Thereby, theX-ray diffraction also confirms that the synthesis and depositionprocesses developed avoided the degradation of molybdenum cluster-basedmaterial. The robustness of the prepared cluster materials is an advantagefor preparing gas sensors and conducting the resistive and opticalmeasurements described below. Regarding the binding interaction betweenthe crystalline cluster phase and the graphene sheets, we think thatit could mainly be associated with supramolecular interactions, suchas Van der Waals and charge electrostatic and polar interactions,due to the polarity induced by the cluster units and the presenceof water molecules embedded into the hydrogen-bonded network of thecrystalline cluster phase and the preference of such water moleculesto adsorb on residual defects and sites with oxygen-containing functionalgroups.36 However, thecluster immobilization of {Mo6Ii8}4+ cluster cores through coordinative bonds to such oxygenfunctionalities, as occurs with GO, cannot be discarded.29 Additional research on the study of the bindinginteractions involved is under current investigation.The crystalline cluster sites are preferentially exposed to gas adsorption, thesensing is probably promoted by hydrogen bonding between the apicalcluster ligand (hydroxo or water) and the gaseous analytes. To revealthe interactions between the [Mo6Ii8(OH)a4(H2O)a2] clusters and target molecules, DFT calculations were performedusing the BPE0 functional because of its robustness and excellentperformance for treating hydrogen bonds.56 Two stereoisomers, namely trans and cis-[Mo6Ii8(OH)a4(H2O)a2], were built as representativemodels for adsorption sites, and the reliability of this functionalwas confirmed by the analysis of the computed geometries of the isomers,which shows a good fit to the X-ray diffraction structure . The energetic interactions betweenthe cluster compound and the gas molecules were studied by computingthe binding energies (BEs) associated with the formation of a cluster-gasmolecule (NH3 or NO2) adduct in a 1:1 ratio.In all the adducts, the gas interacts directly with water and hydroxoligands (S13) by hydrogen bonding in most cases, withBEs in good agreement to H-bonding energies (Table S1). ca. −18 and −7kcal/mol, associated with the isomers with H2N···HOHand NO2···HOH interactions, respectively.The gas-cluster adducts become energetically more unstable when thehydroxo ligand is involved (Table S1).In all cases, BEs are negative, indicating that the formation of cluster-gasmolecule adducts is exothermic. Thus, NH3 and NO2 molecules are likely to be adsorbed onto the surface of the cluster-basedcrystalline material.The factor responsiblefor this orthogonal behavior is related to the interaction betweenthe target gas analytes and the molybdenum cluster-based material.The integrity of the Moligands 7 and S13tructure S12. The 6Ii8(OH)a4(H2O)a2] toward NH3 andNO2 under resistive and optical conditions is representedin 2 would favor the increase of positive charges in graphenelayers, resulting in a major chemoresistive response. In the presenceof NH3, its electron donor ability practically does notinfluence the electronic characteristics of the clusters, and, asa consequence, they would remain intact to exchange charges with graphene.This could be associated with the difficulty in the reduction of the{Mo6Ii8}4+ cluster corecompounds, considering their intrinsic redox properties.57 One question remains open regarding the smallchemoresistive response of Mo6@graphene in the presenceof ammonia in comparison with one of the bare graphenes 30 would promotethe transfer of some holes from the graphene layer to the metalliccluster and concomitant compensation of charges, giving rise to adiminution of the resistive response.A tentative description of the sensing mechanismof . This was prepared by properlyadapting the synthetic procedures reported in the literature fromthe (Bu4N)2[Mo6Ii8Ia6] and silver acetate materials.61 In a typical procedure for preparationof Mo6, the cluster precursor (30 mg) was dispersed ina mixture (15 mL) of water, acetone, and triethylamine in 50/45/5%v/v, respectively, in a round-bottom flask with continuous magneticstirring (500 rpm) at room temperature. Afterward, a bubbler was connectedto the flask, and the resulting yellow-orange suspension was heatedto 35 °C for 1 h. Over time, the mixture evolved to a solution,which was added into a 10 mL vial sealed with a holey parafilm forits slow evaporation for 5–7 days. Finally, red microcrystalswere collected from the remaining solution. The crystalline materialwas washed twice with acetone and once with Milli-Q water (10 mL).Notably, the optimum crystal size is achieved when the solution becomestransparent.The synthesis of Mo2/g,Strem Chemicals Inc., USA). Then, the resulting suspension was placedin an ultrasonic tip for achieving a proper graphene exfoliation.Particularly, a pulsed sonication (1s on/2s off) at 280 W for 90 minwas applied. A 1 mL suspension of Mo6 (0.25 mg/mL) in dichloromethanewas prepared by sonication for 30 min in an ultrasonic bath. Afterward,the Mo6 mixture was added to the graphene suspension, resultingin graphene loaded with a 5% wt of the Mo6 cluster. Themixture was homogenized for 10 min under stirring. Subsequently, thesolution was sonicated in an ultrasonic bath for 1 h, resulting ina suitable distribution of molybdenum clusters supported on graphene(Mo6@Graphene). For the characterization of the nanomaterial,the resulting suspension was deposited onto appropriate wafers directedto each characterization technique and placed onto a hotplate at amoderate temperature (60 °C). For sensing studies, the suspensionwas deposited via spray coating on alumina substrates comprising platinumscreen-printed electrodes .First, the substrates were placed onto a hotplate at a moderate temperature(60 °C), and then, the deposition process was started after fewminutes for ensuring the correct heating up of the substrate. Thespray coating deposition was performed using nitrogen as the carriergas for about 45 s. As a result, homogeneous sensitive films wereobtained with an average thickness of 1 μm  and optical profilometermeasurement ).For supporting the molybdenum clusterson graphene, two solutionswere prepared in parallel. First, a 10 mL graphene suspension (0.5mg/mL) in dichloromethane was performed employing graphene nanoplateletaggregates . FESEM imageswere recorded with a Zeiss Ultra 55 field FESEM apparatus  equipped with a BSE detector. Samples for HR-TEM were depositedonto carbon-coated copper grids. HR-TEM images were recorded by usinga JEOL JEM2100F microscope operating at 200 kV. The molybdenum andiodine contents of the Mo6@Graphene sample were determinedby dark-field scanning transmission electron microscopy (STEM) EDSanalysis conducted by using an EDAX system (Oxford Instruments) attachedto a JEOL JEM2100F electronic microscope. The thickness of the filmwas measured with a MicroXAM-100 3D surface profilometer. The cross-sectionwas examined by FESEM (Zeiss Ultra 55 instrument).UV–vis spectra wereacquired at 20 °C, employing a Varian Cary 50 Conc spectrophotometerequipped with 10 × 10 mm quartz cells. Steady-state PL measurementswere recorded in an Edinburgh Instruments FLS1100 spectrofluorometerusing a 450 W xenon lamp light equipped with a double monochromatorfor excitation and emission coupled to a cooled photomultiplier (PMT-980).The Raman spectra were obtained from solid samples previously depositedonto quartz substrates using a “Reflex” Renishaw spectrometerequipped with an Olympus microscope. The exciting wavelength was 514nm of an ArFigure S14), thegas sensors were placed in an airtight testing chamber (dead volumeof 35 cm3). The chamber was subsequently connected to agas mixing and delivery system comprising different calibrated gascylinders. A pure dry air atmosphere (air premier purity: 99.999%)was employed during the sensing test and used as a carrier gas forthe target gases. It is worth highlighting that a residual amountof relative humidity (3–4%) is present in dry conditions. Theoverall flow was adjusted at a low rate (100 mL/min) using a set ofmass-flow controllers and electro-valves. Since it is well known that higher flow rateslead to greater resistance changes, it is worth noting that the lowflow rate applied sacrifices response intensity but has the advantageof enabling lower power consumption and working under more realisticexperimental conditions. The effect of the ambient moisture on thesensor’s performance was also assessed by using a controllerevaporator mixer  forhumidifying the atmosphere.Once homogeneouslayers were achieved (R/R0) expressedin percentage. Where R0 is defined as the resistance levelof the sensor in the air, while ΔR correspondsto the resistance changes obtained over 5 min of gas exposure.The sensor resistances were monitoredusing a multimeter , registering the resistancechanges induced by different concentrations of gases. Specifically,the sensors were exposed to given concentrations of target gases for5 min and subsequently stabilized for 15 min under dry airflow. Severalconcentrations of gas species were applied by performing successivedilutions with pure dry air, and the sensing responses were definedas (Δ6 or Mo6@Graphene) in 1mL of acetonitrile and sonicated for 10 min. Subsequently, the suspensionswere deposited by drop-casting on an acid-treated quartz substrate(Mo6 sample) and an alumina substrate (Mo6@Graphenesample) placed on a hotplate at 50 °C. Then, the samples wereplaced inside a screw cap cuvette and fixed with a small piece ofcommercial Blu-Tack . The sampleswere purged for 20 min with Ar, air, and NO2  before each in situ measurement. The measurementswith NH3 were done by gas diffusion of 10 μL of ammoniumhydroxide solution  placed atthe bottom of the cuvette. The amount of gaseous NH3 wascalculated from Henry’s law (Hcp = 0.58 M/Pa at298.15 K) considering the equilibrium concentration of ammonia insolution (kb = 1.8 × 10–5).Samples for carryingout UV–vis and PL experiments were prepared by dispersing 5mg of the sample  before and after exposureto NO63 Relativisticelectron core potentials from the Stuttgart group and its associatedbasis sets64 were used to represent themolybdenum65 and iodine atoms66 and augmented in the case of Mo by an f polarizationfunction (Mo/α = 1.043)67 and inthe case of I, with a d polarization function (I/α = 0.289).68 The 6-31G basis set was used to representthe remaining atoms  of the molecular systems. The geometryoptimizations were performed in the gas phase without any symmetryconstraint, followed by analytical frequency calculations to confirmthat a minimum has been reached. Zero-point energies were includedin BE calculations, and the BEs were calculated using BE = Ecluster-molecule – [Ecluster + Emolecule].The calculationswere conductedwith the Gaussian 09 program suite. Density functional theory wasapplied with the PBE0 functional.trans- and cis-[Mo6Ii8(OH)a4(H2O)a2] isomers were built as representativemodels for adsorption sites due to the fact that water and hydroxopositioning around the octahedral metal framework cannot be inferredfrom the X-ray diffraction structure. The guess geometries of thegas–cluster adducts were raised considering a random positioningof the gas analyte with respect to all the cluster ligands  of the two cluster isomers.The


Trichilia dregeana Sond are traditionally used to treat wounds. Even though there have been in vitro studies and claims supporting wound healing, there are no scientific data on in vivo wound healing and anti-inflammatory activities of the leaves of T. dregeana.  The leaves of T. dregeana in mice.  This study aimed to evaluate wound healing and anti-inflammatory activity of 80% methanol crude extract and solvent fractions of T. dregeana were dried, ground, and macerated with 80% methanol three times successively. The crude extract was fractioned by water, ethyl acetate, and hexane separately. The acute dermal and oral toxicity tests were done by applying 2000 mg/kg of 10% (w/w) crude extract ointment (CEO) topically and 2000 mg/kg of crude extract orally, respectively. The wound healing activity of the crude extract was evaluated on excision, incision, and burn wound models while the fractions were evaluated only on excision wound model. The anti-inflammatory activity of the crude extract was evaluated on xylene-induced ear edema and cotton pellet granuloma tests.  The leaves of p < 0.001) wound contraction and period of epithelialization from day 4 onwards as compared to simple ointment (SO) on both excision and burn wounds. The tensile strength was increased significantly (p < 0.001) for the CEO-treated mice as compared to both untreated and SO groups. The crude extract also showed anti-inflammatory activity at 100, 200, and 400 mg/kg by inhibiting ear edema, exudate, and granuloma formation as compared to the SO group.  Both acute dermal and oral toxicity tests were found to be safe after topical application of 2000 mg/kg of 10% (w/w) CEO and oral administration of 2000 mg/kg of crude extract suspension, respectively. Both 5% and 10% (w/w) CEO produced significant (T. dregeana for wound healing. The increase in wound contraction, reduction in period of epithelialization, and increase in tensile strength support the traditional claims of  Wound is defined as damage to the integrity of biological tissues, including skin, mucous membranes, and organ tissues, due to various types of trauma . The losTrichilia dregeana Sond which is commonly known as Cape Mahogany and Forest Natal Mahogany in South Africa is a large, up to 35 m in height and 1.8 m in diameter, evergreen tree which inhabits forests in high rainfall areas. It has beautiful dark foliage and a large rounded crown from the Meliaceae family [e family  , hard paraffin , white soft paraffin (Queens Hygenic Industries plc), cetostearyl alcohol , methanol absolute , n-hexane , ethyl acetate , distilled water , nitrofurazone skin ointment 0.2% , ketamine hydrochloride injection USP , diazepam injection , 70% alcohol , normal saline 0.9% , bees wax , formalin 10% , hematoxylin , eosin , Glacial acetic acid , potassium iodide , ammonia solution , mercuric chloride , iron chloride , lead acetate , trihydrate, and Tween 80  were procured from legal suppliers as per the required standard and analytic grade.Sensitive digital weighing balance , lyophilizer, rotary evaporator , dry oven , light, vacuum pump, desiccator, deep freezer, water bath, mortar and pestle, ointment slab, sharp sterilized scissors, surgical threads with curved needles, forceps, surgical scalpel blade, Erlenmeyer conical flask, Whatman filter paper (No 1) , beaker, Buchner funnel, syringe with needle, glove, cotton swab adhesive plaster zinc oxide, face mask, head cover, gauze, gloves, cotton swab, permanent marker, and transparent polythene graph paper were used.Trichilia dregeana Sond leaves were collected in early March 2021 from Yebbu town, Jimma Zone, Ethiopia. It was identified and authenticated by Mr. Wondie Mebrat, Department of Biology, College of Natural and Computational Science, Debre Tabor University, and a voucher specimen was deposited with collection number DG001.The fresh Healthy, either sex, adults, 20–30 grams of weight, and 6–8 weeks of age Swiss albino mice were obtained from animal house facilities of the Department of Pharmacology, University of Gondar. The mice were caged individually at room temperature and under 12 hours of light and dark cycles with a standard pellet diet and free water access  and alloThe debris from the collected plant materials was rinsed with tap water and subjected to shade drying. The dried plant materials were ground to a coarse powder using mortar and pestle and stored in an air-tight container until extraction was started.A total of 2.5 kg powder was macerated in 15 L of 80% methanol (1 : 6 w/v) in round bottom flask with intermittent shaking for three days. On the third day, the extract was filtered using surgical gauze and Whatman filter paper number 1 consecutively. The marc was re-macerated twice in the same manner, and the filtrates were collected and evaporated using a rotary evaporator set at 40°C, and concentrated aqueous solution remained. The remaining concentrated aqueous solution was further evaporated using a dry oven at 40°C. By using a deep freezer, the concentrated filtrate was frozen overnight at −40°C, and then the frozen filtrate was lyophilized at vacuum pressure 0.200 mBar and −40°C to remove water. The dried powder was weighted, packed in an air-tight container, and stored at 4°C temperature. The yield obtained after extraction of dried powder material was 182.25 g (7.29%).For fractionation, a total of 115 g of the crude extract was suspended in 690 ml of distilled water (1 : 6 w/v) using a separatory funnel. Then, the same amount of n-hexane was added, mixed well, and allowed to settle and form distinct layers. Once the distinct layers were formed, n-hexane fraction was separated by eluting the bottom aqueous layer from the separatory funnel. This step was repeated two more times, and the n-hexane fractions were collected in the same container. To get ethyl acetate fractions, an equal amount of ethyl acetate to that of distilled water was added to the aqueous fraction. The ethyl acetate fraction was separated after a distinct layer was formed between ethyl acetate and aqueous fraction by collecting the bottom aqueous layer. This process was also repeated two more times, and ethyl acetate fractions were collected in the same container. The fractions of all n-hexane and ethyl acetate were evaporated using a rotary evaporator while aqueous fraction was concentrated using an oven at 40°C before undergoing lyophilization at vacuum pressure 0.200 mBar and −40°C. The percent yields of the dried fractions were 33.75%, 6.63%, and 56.21% for n-hexane, ethyl acetate, and aqueous fractions, respectively. The fractions were stored at 4°C in the refrigerator .Using the formula described in British Pharmacopeia , the simAll ingredients were weighted properly. Based on their descending order of melting points which are hard paraffin, cetostearyl alcohol, wool fat, and white soft paraffin, all ingredients were placed and added to the evaporating dish and melted over a water bath. The mixture was stirred continuously to ensure homogeneity . This siT. dregeana leaves was screened based on standard procedures [The presence of alkaloids, saponins, flavonoids, phenols, steroids, glycosides, tannins, anthraquinones, and terpenoids in the crude extract as well as n-hexane, ethyl acetate, and aqueous fractions of ocedures .Acute oral toxicity test was carried out based on OECD 425: “Limit test at 2000 mg/kg” [An acute dermal toxicity test of 80% methanol crude extract of the plant was carried out and evaluated according to OECD 402 and 404 guidelines , 22. FivTrichilia dregeana Sond leaves, a total of 186 mice were used. To evaluate the wound healing activity of the extract, 24 mice were grouped into four groups for each excision and burn wound model while 30 mice were grouped into five for the incision wound model. Each group contains six mice. In all three models, group I was treated with simple ointment (serve as negative control), groups II and III were treated by 5% and 10% (w/w) extract ointment, respectively. Group VI was treated with nitrofurazone 0.2% w/v ointment (served as positive control). Group V of incision wound model was left untreated (served as an untreated control group).For evaluation of wound healing and anti-inflammatory activities of Solvent fractions were evaluated on an excision wound model using eight groups (each has six mice). Group I was treated with simple ointment. Groups II and III were treated with 5% (w/w) and 10% (w/w) aqueous fraction (AQF) ointment formulations, respectively. Groups IV and V were treated with 5% (w/w) and 10% (w/w) ethyl acetate fraction (EAF) ointment formulations, respectively. Groups VI and VII were treated with 5% (w/w) and 10% (w/w) n-hexane fraction (NHF) ointment formulations, respectively. Group VIII was treated with 0.2% (w/v) nitrofurazone ointment.To evaluate the anti-inflammatory activity of the extract, five groups (each containing six mice) were used for each of xylene-induced ear edema model and cotton pellet-induced granuloma model. Group I was treated with 2% Tween 80 at a dose of 10 ml/kg (served as negative control). Groups II, III, and IV were treated with 100 mg/kg, 200 mg/kg, and 400 mg/kg extract, respectively. Group V was treated with indomethacin 10 mg/kg (served as positive control). At the end of the experiment, each mouse was euthanized by using high doses of ketamine  and diazepam. Finally, the remnants were buried in a proper area to avoid environmental contamination.2 circular area and 2 mm depth was created using forceps and scissors  and left undressed. From day 1, the ointments were applied once daily for 9 days. On post-wounding day 8, the suture was removed and the extent of healing was assessed by measuring the tensile strength using continuous water flow method on post-wounding day 10. Two forceps were inflexibly applied at 3 mm away from the edge of the wound facing each other on the opposite sides of the incision wound to the anesthetized mice on the operating table. One of the forceps was secure on stands while the other was connected to a freely suspended lightweight plastic of volume 500 ml into which the water flowed continuously and gently. The water flow was stopped at the moment the wound was opened up and the volume of the collected water in the reservoir was recorded as tensile strength . The bre(1) Burn Wound Model. Twenty-four mice were anesthetized, fur from the back was shaved, and they were decontaminated similarly to that of excision and incision models. A circular cylinder of 300 mm2 opening into which the molten wax at 80°C was poured in was placed on the shaven part of the mice for 10–12 minutes until the poured hot molten wax gets solidified, and then the wax was removed. This created a partial-thickness circular burn on the area. Then, the mice were placed back in their cage individually. Treatments were applied over the wounded area daily until the wound is epithelialized. The healing progress was assessed every 2 days, and the percentage of wound contraction was calculated, period of epithelialization was recorded, and histopathological analysis was performed [erformed .(1) Xylene-Induced Ear Edema Model. The methods previously used by Manouze et al. [e et al.  were fole et al. .(3)Ear s(2) Cotton Pellet-Induced Granuloma. A chronic anti-inflammatory effect of 80% methanol extract of the plant was carried out according to previously used methods by Gou et al. [u et al.  with somu et al. . The disPercentage protection from granuloma development was calculated using the following formula:p < 0.05 was considered statistically significant.The results of the experiment were expressed as mean ± standard error of the mean, and SPSS version 26 was used to analyze the results. One-way analysis of variance (ANOVA) was used for the test of statistical significance followed by Tukey's post hoc test. Phytochemical screening of 80% methanol crude extract and solvent fractions showed the presence of different secondary metabolites .No sign of toxicity was observed after oral administration of 2000 mg/kg of the extract as well as dermal application of 2000 mg/kg of the 10% (w/w) extract ointment within the first 24 and 48 hours, and no death was registered after 14 days of follow-up. The result suggests that oral LD50 of the plant extract is greater than 2000 mg/kg in mice.(1) Healing Activity of the Crude Extract. Treatment with 10% (w/w) extract ointment showed significant wound contraction (p < 0.01) on day 2 and on other post-wounding days (p < 0.001) as compared to simple ointment. Similarly, treatment with 5% (w/w) extract ointment showed significant wound contraction on all post-wounding days, on day 2 (p < 0.05), and on the rest of the post-wounding days (p < 0.001) as compared to simple ointment.p < 0.001) and day 6 (p < 0.01) as compared to 5% (w/w) extract ointment. Treatment with 10% (w/w) extract ointment also showed better wound contraction on most post-wounding days as compared to 0.2% nitrofurazone ointment though the differences were insignificant (Treatment with both 10% (w/w) extract and 0.2% nitrofurazone ointments showed significant wound contraction on day 4 (nificant .Complete percentage of wound contraction for groups treated with 10% (w/w) extract ointment, 5% (w/w) extract ointment, 0.2% (w/v) nitrofurazone ointment, and simple ointment was observed on day 16, 18, 18, and beyond, respectively Figures  and 5.p < 0.001) short period of epithelialization as compared to simple ointment. Mean period of epithelialization in mice treated with 5% (w/w) extract ointment, 10% (w/w) extract ointment, and 0.2% (w/v) nitrofurazone ointment has been reduced by 24.99%, 26.54%, and 25.79%, respectively, as compared to simple ointment (Treatment with both 5% (w/w) and 10% (w/w) extract ointment as well as 0.2% (w/v) nitrofurazone ointment showed significantly (ointment .p < 0.001) wound contraction on all post-wounding days as compared to all simple, 5% (w/w) ethyl acetate fraction, 10% (w/w) ethyl acetate fraction, 5% (w/w) hexane fraction, and 10% (w/w) hexane fraction ointments. Similarly, 5% (w/w) aqueous fraction ointment showed significant wound contraction (p < 0.001) as compared to simple ointment (p < 0.01) as compared to all 5% (w/w) ethyl acetate fraction, 10% ethyl acetate fraction, 5% (w/w) hexane fraction, and 10% (w/w) hexane fraction ointments on post-wounding day 2. From post-wounding day 4 onwards, 5% (w/w) aqueous fraction ointments showed highly significant (p < 0.001) wound contraction when compared to all simple, 5% (w/w) ethyl acetate fraction, 10% (w/w) ethyl acetate fraction, 5% (w/w) hexane fraction, and 10% (w/w) hexane fraction ointments.Treatment with 10% (w/w) aqueous fraction ointment showed significant (p < 0.05) and from day 14 onwards (p < 0.01) as compared to simple ointment, while 10% (w/w) ethyl acetate fraction ointment showed significant wound contraction from day 6 to 8 (p < 0.05), from day 10 to 12 (p < 0.01), and from day 14 onwards (p < 0.001) as compared to simple ointment.Treatment with 5% (w/w) ethyl acetate fraction ointment showed significant contraction from day 10 to 12 (p < 0.05) and from day 12 onwards (p < 0.01) as compared to simple ointment, while 10% (w/w) hexane fraction ointment showed significant wound contraction from day 4 to 8 (p < 0.05), from day 10 to 12 (p < 0.01), and from day 14 onwards (p < 0.001) as compared to simple ointment. Nitrofurazone also showed significant wound contraction (p < 0.001) on all post-wounding days as compared to simple ointment (Treatment with 5% (w/w) hexane fraction ointment showed significant contraction on day 10 (ointment .Mice treated with 10% (w/w) aqueous fractions ointment showed complete wound closure by day 16 while it took beyond day 16 for complete wound closure by other fraction ointments Figures .p < 0.001) as compared to all simple, 5% (w/w) ethyl acetate fraction, and 5% (w/w) hexane fraction ointments. It also showed significantly short period of epithelialization (p < 0.01) as compared to both 10% (w/w) ethyl acetate fraction and 10% (w/w) hexane fraction ointments. Treatment with 10% (w/w) aqueous fraction ointment reduced period of epithelialization significantly (p < 0.001) when compared to all simple, 5% (w/w) ethyl acetate fraction, 10% (w/w) ethyl acetate fraction, 5% (w/w) hexane fraction, and 10% (w/w) hexane fraction ointments.Treatment with 5% (w/w) aqueous fraction ointment showed significantly short period of epithelialization  as compared to simple ointment. Similarly, treatments with both 5% and 10% (w/w) hexane fraction ointments also reduced the period of epithelialization significantly  as compared to simple ointment.Groups treated with both 5% and 10% (w/w) ethyl acetate fraction ointments reduced the period of epithelialization significantly (Treatment with 10% (w/w) aqueous fraction ointment showed the highest percentage of reduction in the period of epithelialization, 20.85%; on the contrary, treatment with 5% (w/w) ethyl acetate fraction ointment showed the lowest percentage, 6.65%, as compared to simple ointment .p < 0.001) increases in breaking resistance as compared to both simple ointments and left untreated groups. Treatment with 10% (w/w) extract ointment showed the highest percentage of tensile strength, 74.00%, while the simple ointment group showed the least percentage of tensile strength, 12.56%, as compared to left untreated group (Groups treated with 5% and 10% (w/w) extract ointment showed significant (ed group .(1) Wound Contraction. Mice treated with 10% (w/w) extract ointment showed significant wound contraction on day 2 (p < 0.05), on day 4 (p < 0.01), and on the rest of the post-wounding days (p < 0.001) as compared to simple ointment. Similarly, mice treated with 5% (w/w) extract ointment showed significant wound contraction on day 4 (p < 0.05) and on other post-wounding days (p < 0.001) as compared to simple ointment.p < 0.05) and day 8 (p < 0.01) as compared to 5% (w/w) extract ointment. Similarly, treatment with 0.2% (w/v) nitrofurazone ointment showed significant contraction on day 8 (p < 0.05) as compared to 5% (w/w) extract ointment (Treatment with 10% (w/w) extract ointment showed significant wound contraction on day 6 (ointment .Complete wound closure after treatment with 5% (w/w) extract ointment, 10% (w/w) extract ointment, 0.2% (w/v) nitrofurazone ointment, and simple ointment was observed on day 22, 20, 22, and beyond, respectively Figures  and 9.p < 0.001) short period of epithelialization as compared to simple ointment. Mice treated with 10% (w/w) extract ointment also showed significantly (p < 0.05) short period of epithelialization when compared to 5% (w/w) extract ointment.Treatment with 5% and 10% (w/w) extract ointments showed significantly (Treatment with 5% (w/w) extract ointment, 10% (w/w) extract ointment, and 0.2% (w/v) nitrofurazone ointment reduced period of epithelialization by 11.36%, 17.74%, and 14.17%, respectively, when compared to simple ointment .Wound treated with 10% (w/w) extract ointments showed high fibroblast proliferation, collagen deposition, and neovascularization as well as few numbers of inflammatory cells while 5% (w/w) extract ointment-treated wound showed high fibroblast proliferation, moderate collagen deposition, and neovascularization. Nitrofurazone-treated wound showed moderate fibroblast proliferation, collagen deposition, and neovascularization . The hisMice treated with 5% (w/w) aqueous fraction ointment showed high fibroblast proliferation, moderate collagen deposition, and high neovascularization while treatment with 10% (w/w) aqueous fraction showed high fibroblast proliferation, collagen deposition, and neovascularization. Groups treated with 5% (w/w) ethyl acetate fraction, 10% (w/w) ethyl acetate fraction, 5% (w/w) hexane fraction, and 10% (w/w) hexane fraction ointments showed moderate to high number of inflammatory cells, low fibroblast proliferation, invisible to low collagen deposition, and low neovascularization .Treatment with 10% (w/w) extract ointments showed high fibroblast proliferation and collagen deposition while 5% (w/w) extract ointment showed moderate fibroblast proliferation and collagen deposition. Treatment with 10% (w/w) and 5% (w/w) extract ointments also showed moderate and low neovascularization, respectively . As showp < 0.001) at 100, 200, and 400 mg/kg as compared to 2% Tween 80. Treatment with 400 mg/kg also showed significant (p < 0.001) ear edema inhibition as compared to both 100 and 200 mg/kg. Similarly, indomethacin-treated mice showed significant (p < 0.001) inhibition in ear edema as compared to all groups treated with 100 mg/kg, 200 mg/kg, and 400 mg/kg as well 2% Tween 80.The extract inhibited ear edema significantly  inhibits both wet weight (exudate) and dry weight (granuloma) of the cotton pellet as compared to 2% Tween 80. Similarly, treatment with extract at 200 mg/kg and 400 mg/kg significantly (p < 0.001) inhibits the wet weight of the cotton pellet when compared to 100 mg/kg.At 100 mg/kg, 200 mg/kg, and 400 mg/kg, the extract significantly  as compared to 100 mg/kg. Treatment with 400 mg/kg also significantly reduces (p < 0.001) both wet and dry weight of the cotton pellet formation when compared to 200 mg/kg.The extract at 200 mg/kg and 400 mg/kg showed a significant reduction in dry cotton pellet weight (p < 0.001) inhibition in exudative deposits when compared to 100 mg/kg, 200 mg/kg, and 400 mg/kg as well as 2% Tween 80. Similarly, indomethacin at 10 mg/kg showed significant inhibition (p < 0.001) in proliferative deposits when compared to all 200 mg/kg, 100 mg/kg, and 2% Tween 80 as well as 400 mg/kg (p < 0.01).Indomethacin at 10 mg/kg showed significant  wound contraction in excision wound model. The percent of wound contraction produced by 10% (w/w) extract ointment on day 14 (98.25%) is comparable to that of Becium grandiflorum Lam which showed 100% contraction by day 14 [Treatment with 5% and 10% (w/w) extract ointment showed significant  of albicans , 40. Bacalbicans . Antimic healing .In the excision wound model of solvent fractions, the aqueous fractions showed a better effect. This difference could be related to either the accumulation of the majority of secondary metabolites in an aqueous fraction . High fiTrichilia dregeana Sond leaf crude extracts could be due to the plant extract's role in promoting collagen synthesis, maturation, and stabilization.The crude extract was also evaluated for wound healing activity in incision wounds by measuring the tensile strength, the resistance of the tissue to break under external force. The significant resistance to breaking might be related to the quality of the repaired tissue which is mainly determined by the regeneration of collagen. Collagen gives strength and integrity to the healed wound that helps the cells to adhere together and resist breaking tension . This siTrichilia dregeana Sond leaves possess antioxidant, antimicrobial, and anti-inflammatory activity [Wound healing activity of the plant extract was also evaluated in the partial-thickness burn wound model in which it showed significant contraction. The creation of partial-thickness burn wound forms three zones which are zone of necrosis at the area of applications, zone of hypoperfusion in the area surrounding the zone of necrosis, and zone of inflammation just next to a zone of hypoperfusion. Microbial colonization, persistent hypoperfusion, excessive free radical generation, and severe inflammation advance the partial-thickness wound to full-thickness wound which delays the healing and makes it more complicated . In prevactivity , 39, 40.Trichilia dregeana Sond leaf extracts contain alkaloids, phenols, tannins, flavonoids, anthraquinones, terpenoids, steroids, and saponins as evidenced by phytochemical tests. This finding is consistent with the phytochemical content of Trichilia emetica Vahl, a plant from the same genus [me genus . Differeme genus .Trichilia dregeana Sond leaf extracts significantly inhibited ear edema formation in the xylene-induced ear edema model. This suggests the plant extract has activity against acute inflammation. This anti-inflammatory activity of the plant is in line with in vitro tests done previously which showed that different solvent extracts of Trichilia dregeana Sond leaves possess anti-inflammatory activity by inhibiting COX 1 and 2 [ 1 and 2 , 40, 47. 1 and 2 .Achyranthes aspera L. [In the cotton-induced granuloma formation test model, the extract significantly inhibited exudate and granuloma formation which is comparable to the effect shown by spera L. . These aspera L. . ExudateThe extract also inhibits the formation of granuloma significantly. More monocytes were drawn to the area, where they joined to create granulomas around the foreign body that were multinucleated giant cells . ProstagT. dregeana showed wound healing activity by increasing wound contraction, decreasing period of epithelialization, and increasing resistance to breakage in different wound models. Besides, the plant extract also showed anti-inflammatory activity by inhibiting ear edema formation, exudate formation, and granuloma formation in different inflammation models. The aqueous fraction showed wound-healing activity by accelerating wound contraction and shortening the period of epithelialization in the excision wound model, which supports the conventional uses in which traditional healers first soak the plant in water before applying it to the wound.Eighty percent methanol extract of