Amyloid plaques contain many proteins in addition to beta amyloid (Aβ). Previous studies examining plaque-associated proteins have shown these additional proteins are important; they provide insight into the factors that drive amyloid plaque development and are potential biomarkers or therapeutic targets for Alzheimer’s disease (AD). The aim of this study was to comprehensively identify proteins that are enriched in amyloid plaques using unbiased proteomics in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. We focused our study on early onset AD as the drivers of the more aggressive pathology development in these cases is unknown and it is unclear whether amyloid-plaque enriched proteins differ between subtypes of early onset AD. Amyloid plaques and neighbouring non-plaque tissue were microdissected from human brain sections using laser capture microdissection and label-free LC–MS was used to quantify the proteins present. 48 proteins were consistently enriched in amyloid plaques in EOAD and DS. Many of these proteins were more significantly enriched in amyloid plaques than Aβ. The most enriched proteins in amyloid plaques in both EOAD and DS were: COL25A1, SMOC1, MDK, NTN1, OLFML3 and HTRA1. Endosomal/lysosomal proteins were particularly highly enriched in amyloid plaques. Fluorescent immunohistochemistry was used to validate the enrichment of four proteins in amyloid plaques  and to compare the amount of total Aβ, Aβ40, Aβ42, phosphorylated Aβ, pyroglutamate Aβ species and oligomeric species in EOAD and DS. These studies showed that phosphorylated Aβ, pyroglutamate Aβ species and SMOC1 were significantly higher in DS plaques, while oligomers were significantly higher in EOAD. Overall, we observed that amyloid plaques in EOAD and DS largely contained the same proteins, however the amount of enrichment of some proteins was different in EOAD and DS. Our study highlights the significant enrichment of many proteins in amyloid plaques, many of which may be potential therapeutic targets and/or biomarkers for AD.The online version contains supplementary material available at 10.1186/s40478-022-01356-1. Amyloid plaques are a neuropathological hallmark of Alzheimer’s disease and primarily consist of the protein beta amyloid (Aβ). However, it is often overlooked that amyloid plaques also contain hundreds of proteins in addition to Aβ. These include proteins that directly interact with Aβ (e.g. apolipoprotein E ), proteiPrevious studies have typically used immunohistochemistry to identify amyloid plaque proteins. Mass spectrometry-based proteomics is an alternative approach that allows efficient quantification of thousands of amyloid plaque proteins simultaneously. Proteomics also offers additional advantages of allowing discovery of novel plaque proteins due to its unbiased nature and bypassing complications due to antibody sensitivity and specificity issues. Given these significant advantages, we recently developed a localized proteomics approach to analyze the proteome of neuropathological lesions in AD such as plaques and neurofibrillary tangles –13.The significant heterogeneity in the clinical and neuropathological phenotype of AD suggests that multiple subtypes of AD exist. Previous studies have used various approaches to define AD subtypes –17. SomeAPP, PSEN1 and PSEN2 mutations. The cause of the remaining ~ 90% of EOAD cases is unknown and these cases are therefore characterized as sporadic EOAD. It is currently unclear if the same molecular mechanisms drive sporadic EOAD cases and late-onset AD [The aim of this study was to compare the amyloid plaque proteome in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. Between 5 and 10% of AD cases are considered early onset . Of thesonset AD . DS withonset AD . People onset AD . Intraneonset AD , which ionset AD , 23. Matonset AD . The proonset AD , albeit onset AD  and a hionset AD . Multiplonset AD –31, howeHere, we show that amyloid plaques in DS and EOAD are enriched in many proteins besides Aβ including a common core group of 48 proteins that are enriched in plaques in both AD subtypes. While similar proteins were enriched in both DS and EOAD, some proteins were enriched to a greater extent in plaques in a particular subtype of AD, providing new evidence that some distinctions in plaque protein composition are present.All procedures were performed under protocols approved by the Institutional Review Board at New York University Alzheimer Disease Center, NY, USA. In all cases, written informed consent for research was obtained from the patient or legal guardian, and the material used had appropriate ethical approval for use in this project. All patients’ data and samples were coded and handled according to NIH guidelines to protect patients’ identities.APP, PSEN1 or PSEN2, tissue formalin fixation time < 6 months. Inclusion criteria for DS cases was ABC neuropathological score of A3, B3, C3, formalin fixation time < 6 months. Formalin fixed paraffin embedded tissue blocks containing the hippocampus and surrounding entorhinal/temporal cortex that were collected and processed as part of routine autopsy procedures were used in this study. This region was selected because it contains a high amount of amyloid pathology in EOAD and in DS with AD. N = 3 cases of EOAD, DS, late onset sporadic AD (LOAD) and cognitively normal, age matched controls were included in immunohistochemistry validation studies. Case specific information for the human tissue samples used in this study is included in Table N = 5 cases of early onset sporadic Alzheimer’s disease (EOAD) and n = 5 cases of DS with Alzheimer’s disease were included for proteomic experiments. Inclusion criteria for EOAD included age < 65 years, ABC neuropathological score of A3, B3, C3 , no muta2 solution (Promega) and 4.2 µl Betaine (Sigma). Cycling conditions were at 98 °C for 4 min and 40 cycles at 98 °C/10 s, 63 °C/1 min and 72 °C/1 min 10 s, followed by 72 °C 10 min. All the amplified fragments were resolved on 2% agarose gels, stained with GelRed 10,000X (Biotium) and visualized under UV exposure. DNA was purified from the agarose gel using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (Cytiva) as indicated by the manufacturer, and DNA concentration was quantified using nanodrop One (Thermo Scientific). The second endpoint PCR was performed using the purified DNA with the conditions described previously, except for the concentration of the primers, which was reduced to 0.15 µM. Unpurified PCR products were submitted to Genewiz for Sanger sequencing, and the sequences were analyzed using SnapGene 5.3.1 software  or frozen tissue (FT) for the cases where it was available , Aβ40 , Aβ42 . The peptides were gradient eluted directly into an Orbitrap Fusion Lumos mass spectrometer using a 145-min gradient. The Orbitrap Fusion Lumos mass spectrometer acquired high resolution full MS spectra with a resolution of 240,000 (at m/z 200), AGC target of 1e6, with a maximum ion time of 50 ms, and scan range of 400–1500 m/z. Following each full MS data-dependent low resolution HCD MS/MS spectra were acquired. All MS/MS spectra were collected using the following instrument parameters: rapid ion trap scan rate, ACG target of 2e4, maximum ion time of 150 ms, one microscan, 0.7 m/z isolation window, fixed first mass of 150 m/z and NCE of 32.H. Sapiens proteome database  using the Andromeda search engine [Protein quantitation was performed using MaxQuant software suite v. 1.6.3.4 . Raw dath engine . A list h engine . Missingh engine  using deh engine . To do tp value of p < 0.05 (paired t-test). Fold change difference was selected as the primary determinant of enrichment/depletion in plaques as this correlated best with immunohistochemistry studies, which is the gold standard approach for identifying plaque enriched proteins. Uncorrected p-values were included to provide an indication of variance within a group, however plaque-enriched proteins identified by p-values alone did not correlate as well with prior gold-standard immunohistochemistry studies.Plaque enriched/depleted proteins were determined as those with a fold change difference between plaques and non-plaques > 1.5 fold and an uncorrected t-test and proteins were deemed significantly different based on a combination of p < 0.05 and fold change difference > 1.5.Direct comparison of plaque protein levels in DS and EOAD was performed using plaque protein levels that were normalized to the neighboring non-plaque tissue for each individual case. For this, normalized plaque protein levels were calculated as the ratio of protein intensity in plaques:non-plaques for each case. Differences in normalized plaque protein levels between DS and EOAD were identified using an unpaired General data manipulations and grouping were performed in R v4.0.2  using th. [. was filtered to identify plaque-enriched proteins that were identified by at last 2 peptides, had a fold-change difference of > 1.5 fold between plaques and non-plaques for AD versus controls or preclinical AD versus controls and did not include the word “keratin” or “immunoglobulin” in the protein name to make their data comparable with ours. Proteins with an abundance in the bottom 10% in sAD plaques or preclinical plaques were excluded. Uniprot ID was used to match proteins between studies.Systematic literature searches were used to identify plaque enriched proteins that have been validated in previous studies. A protein was designated a “known plaque protein” if there was published evidence of enrichment in amyloid plaques in human tissue using immunohistochemistry or mass spectrometry. Additional literature searches were used to determine if a protein was functionally associated with either Aβ or APP in instances where there was no immunohistochemistry evidence of presence in plaques. Key words used in these pubmed searches were: “Alzheimer’s and gene ID” or “Alzheimer’s and protein name”. Plaque enriched proteins identified by mass spectrometry were determined by comparison with Xiong et al. , which iChange in brain protein expression in AD versus controls was determined using our in-house developed database—NeuroPro—which combines results from 33 previous studies that used proteomics to identify consistent protein differences between AD and control human brain tissue , 44–73.Proteins were selected for validation studies based on the following criteria: enrichment in both EOAD and DS plaques, protein abundance in the top 50% in amyloid plaques, high fold change enrichment in plaques, appropriate commercial antibody available and limited/no previous evidence of presence in plaques by immunohistochemistry. Based on these criteria the following proteins were selected for immunohistochemistry validation studies: MSN, EZR, SMOC1 and ARL8B. 8 µm formalin-fixed paraffin embedded tissue sections containing the hippocampus and surrounding cortex were used for immunohistochemistry validation studies using the fluorescent immunohistochemistry method described above. Primary antibodies used for these validation studies included: MSN , EZR , SMOC1 , ARL8B , Aβ . The combined formic acid and citrate buffer antigen retrieval method (described above) was used for all validation immunohistochemistry studies. 63× images of fluorescent immunohistochemistry were collected using a confocal microscope Zeiss 700 with the ZEN Black 2.3 SP1 acquisition software. ARL8B immunoreactivity in neurons, microglia and astrocytes was tested using the same method as above with the following primary antibodies: GFAP , IBA1  and MAP2 . A negative control was included in all immunohistochemistry experiments, which consisted of a section of AD hippocampal tissue that underwent the same method with the primary antibody omitted.The percentage of amyloid plaques co-localized with ARL8B or SMOC1 was quantified using whole slide fluorescent scans that were collected using the Aperio VERSA digital slide scanner (Leica) with the 10× objective. Images were visualized and analyzed using the software Aperio ImageScope ver. 12.4.3 (Leica). Plaques co-stained with SMOC1 or ARL8B and Aβ or plaques stained only with Aβ in the hippocampal region were manually counted and then the ratio of co-stained plaques versus total plaques was calculated . The proportion of the plaques was obtained by plotting total number of plaques compared to the plaques co-stained by SMOC1 or ARL8B and Aβ, using the “grouped” layout of GraphPad Prism 8. Significant differences between groups were identified using one-way ANOVA followed by Tukey multiple comparison’s analysis, using GraphPad Prism 8 software.Amyloid plaques in DS and EOAD had similar amounts of total Aβ, Aβ40 and Aβ42  or extracellular proteins (enrichment FDR: 3.34 × 10−8). The enrichment of vesicle proteins was predominantly driven by endosome or lysosome proteins .Examination of the proteins that had the highest enrichment in plaques in both DS and EOAD included many proteins less studied in the AD field Table A, B. TheOur results suggest that that major plaque enriched proteins in EOAD and DS were largely the same. The consistency of protein enrichment in plaques was even noted at an individual case level Fig. C, D. HowWe also directly compared plaque protein levels in DS and EOAD. For this analysis, plaque protein levels that were normalized against background protein levels for each individual case were used. 38 proteins were significantly different between DS and EOAD plaques after correction for background protein differences. 25 proteins were significantly higher and 13 proteins were significantly lower in DS plaques in comparison to EOAD plaques  was selected as it was the most abundant novel plaque protein identified in our study. ARL8B was selected as a representative plaque-enriched lysosomal protein that had no prior immunohistochemistry evidence of presence in amyloid plaques. Moesin (MSN) and SMOC1 were selected as both have only one prior publication confirming their presence in plaques using immunohistochemistry, but no immunohistochemistry evidence of enrichment in plaques in EOAD or DS. Fluorescent immunohistochemistry confirmed that ezrin, moesin, ARL8B and SMOC1 were enriched in amyloid plaques in comparison to surrounding non-plaque tissue in DS, EOAD and late-onset sporadic AD. Moesin Fig. , Ezrin , and SMOSMOC1 strongly co-localized with amyloid fibrils only in a subset of amyloid plaques Fig. . The proARL8B was also abundant in amyloid plaques in all subgroups Fig. . In contWe also validated the presence or absence of these four plaque proteins in vascular amyloid pathology. MSN, EZR and SMOC1 immunoreactivity occasionally co-localized with CAA or in plaques which were in direct contact with blood vessels. However, ARL8B immunoreactivity was absent in vascular amyloid pathology, which is consistent with its weak direct colocalization with Aβ in amyloid plaques Fig. .Fig. 10COur results show that amyloid plaques in DS and EOAD are highly enriched in many proteins in addition to Aβ. Here, we have identified a core group of 48 proteins that are consistently enriched in plaques in comparison to neighboring non-plaque tissue in DS and EOAD. Many of these enriched plaque proteins have been validated in previous studies to colocalize with plaques or correlate with Aβ pathology in typical late onset AD, suggesting that this core group of enriched plaque proteins is consistent in both early and late onset AD subtypes. We also identified 15 novel proteins that were consistently enriched in plaques in both DS and EOAD. Our immunohistochemistry studies showed that while similar proteins are present in plaques in DS and EOAD the relative abundance of some of these proteins  is distinct in plaques in DS and EOAD.Our unbiased proteomics approach highlighted the striking enrichment of many proteins in amyloid plaques that have not been extensively studied in the context of AD such as COL25A1, SMOC1, NTN1, MDK, OLFML3 and HTRA1. The small number of previous studies examining the role of these proteins in AD suggest that these proteins likely have an important role in AD pathology. All of these highly enriched plaque proteins were also enriched in amyloid plaques in typical late onset AD  and 5/6 One of these highly enriched plaque proteins—SMOC1—was the focus of our immunohistochemistry validation studies. The role of SMOC1 in AD and its function in the brain is currently unknown. Single cell RNAseq studies suggest that SMOC1 is enriched in oligodendrocyte precursor cells in the brain  and prevWe hypothesize that the proteins that are highly enriched in amyloid plaques have an important mechanistic role in AD pathology. A common criticism regarding the pathological importance of proteins that accumulate in plaques is that they may not have a mechanistic role in driving pathology and are simply present in plaques by chance. However, a comprehensive review of the literature does not support this criticism. 60% of the 48 proteins commonly enriched in plaques in EOAD and DS have already been confirmed to have a mechanistic role in driving AD pathology in transgenic mouse models or in vitro Table . PreviouThe core group of 48 proteins enriched in plaques in both DS and EOAD were highly enriched in extracellular proteins and endosomal-lysosomal system proteins. The enrichment of extracellular proteins is expected given the extracellular location of amyloid plaques. However, the significant enrichment of endosomal-lysosomal system proteins in plaques was intriguing. A growing body of evidence convincingly shows that Aβ accumulates intraneuronally within endolysosomal vesicles at early stages of AD , 136. EnArl8b (encoded by the gene ARL8B) is an example novel lysosomal protein that we identified as enriched in amyloid plaques in both DS and EOAD. Arl8 is a small GTPase located on lysosomes that facilitates lysosomal trafficking along axons by acting as the linking molecule between lysosomes and kinesin-1 , 143. DiOur immunohistochemistry and literature validation studies showed that amyloid plaque enriched proteins had different colocalization patterns in amyloid plaques. For example, endolysosomal proteins typically have punctate/granular localization in plaques. This staining pattern was observed for ARL8B in our study, which was identical to the staining pattern seen for other lysosomal proteins in past studies such as cathepsin D , cathepsDirect comparison of the amyloid plaque proteome in EOAD and DS showed that amyloid plaques in the two subtypes of younger onset AD had a very similar plaque protein composition. This shows that despite the different disease initiating factors, the resulting amyloid plaques still largely contain the same proteins. While some proteins were enriched to a much greater extent in amyloid plaques in either DS or EOAD (e.g. SMOC1), the trend for enrichment in both subtypes was highly similar. It is still unclear how these relative plaque protein differences influence AD pathogenesis. Future mechanistic studies examining how each of these proteins influence Aβ aggregation or clearance are needed. Future proteomic studies examining whether these major plaque enriched proteins are also enriched in other subtypes of AD  would also potentially provide insight into differences into the rate, topography or type of plaque pathology between these subtypes.In conclusion, we provide a new resource for the AD field that comprehensively characterizes proteins that are enriched in amyloid plaques in multiple subtypes of AD. We propose that these consistently enriched amyloid plaque proteins provide insight into the mechanisms driving amyloid plaque development in AD and are potentially novel drug targets and/or biomarkers for AD.Additional file 1: Table S1. Total imputed dataset. Table S2. Unimputed data. Table S3. Proteins enriched in plaques in both EOAD and DS  Schematic diagram of the APOE genotyping methodology. Six scrolls of 8 µm were sectioned from FFPE blocks and DNA was isolated with the automated QIAsymphony SP. An endpoint PCR was performed, samples were resolved in a 2% agarose gel and amplified DNA was purified from the gel. A second PCR with the purified DNA was performed and un-purified PCR products were sequenced to determine the APOE genotype. (B) Representative gel of EOAD and DS samples used for sequencing. APOE band is located at 348 bp. (C) Sanger sequencing chromatogram showing the nucleotides located in the single-nucleotide polymorphisms (SNPs) rs429358 and rs7412, which determine the APOE variant ε3.


Moreover, it is worth noting that the Rh@Pc catalyst can remain stable against dissolution under the pH = 0 condition. Ab initio molecular dynamic calculations suggest that Rh@Pc could remain stable at 300 K. Our findings highlight a novel family of two-dimensional (2D) materials as efficient and stable SACs and offer a new strategy for catalyst design.Rationally designing efficient, low-cost and stable catalysts toward the oxygen evolution reaction (OER) and the oxygen reduction reaction (ORR) is of significant importance to the development of renewable energy technologies. In this work, we have systematically investigated a series of potentially efficient and stable single late transition metal atom doped phthalocyanines  as single-atom catalysts (SACs) for applications toward the OER and ORR through a computational screening approach. Our calculations indicate that TM atoms can tightly bind with Pc monolayers with high diffusion energy barriers to prevent the isolated atoms from clustering. The interaction strength between intermediates and TM@Pc governs the catalytic activities for the OER and ORR. Among all the considered TM@Pc catalysts, Ir@Pc and Rh@Pc were found to be efficient OER electrocatalysts with overpotentials  ηOER of 0.41 V followed by Rh@Pc with an ηOER = 0.44 V. For the ORR process, the best catalyst is Rh@Pc with an ηORR of 0.44 V followed by Ir@Pc (ηORR = 0.55 V).The best catalyst for the OER is Ir@Pc with an  These electrochemical technologies generally involve the OER and ORR, which have attracted much interest in the energy conversion research. The OER occurs on the anode side of an electrochemical water splitting cell, while as a reverse reaction of the OER, the ORR occurs on the cathode side of a fuel cell and a metal–air battery. Currently, Ru/Ir oxides4,6 and Pt oxides as well as their alloys7–9 are commonly used as the most efficient OER and ORR catalysts, respectively. However, these noble metals are rather expensive, and only a small number of surface sites can serve as catalytically active sites, which make their use rather inefficient. Therefore, the design and development of novel families of low-cost electrocatalysts whose catalytic activities are comparable or even higher than those of noble metal oxides are of significant importance.Increasing energy demand and fast depletion of fossil fuels have led to search for alternative energy sources and efficient energy conversion technologies.10 However, the isolated metal atoms are easy to aggregate to form clusters or nanoparticles due to their high surface energies.11 To maintain the stability of SACs, the previous experimental and theoretical studies have demonstrated that isolated metal atoms can bind at appropriate defects or hollow sites of 2D supporting materials to avoid cluster aggregation.12,13 Moreover, due to the confinement of single atoms in the appropriate supports, the electronic properties of doped single metal atoms can be tuned with different transition metal elements which can enhance their catalytic activity.14–16 In the last few years, single metal electrocatalysts for the OER/ORR have been the subjects of extensive studies. Especially, metal and nitrogen co-doped carbon (M–Nx–C) materials have been demonstrated in many investigations to show OER/ORR performance with comparable activities to those of Ir/Pt oxides,17–21 thus suggesting a promising way to replace noble metal oxides by M–Nx–C catalysts. For example, our previous study22 found that the OER overpotential roughly decreases with the increase of the coordination number of N–TM. Wang et al.23 reported that for Co and N codoped graphene , both OER and ORR overpotentials roughly decrease with the increase of the doping concentration of nitrogen. The root of the M–Nx–C materials used as electrocatalysts can be traced back to the discovery of the capability of M–N4 macrocycle complexes (e.g. porphyrin) toward the ORR.24,25Recently, single-atom catalysts have attracted extensive attention, as metal atoms individually dispersed on supports can be promising for the maximum metal element utilization.26–30 The synthesis procedure of MPc is flexible so that metal atoms can be replaced by other TM atoms.31 More importantly, due to the large surface area, unique atomic structures, and intrinsic properties of dispersed metal sites, MPc monolayers and their derivatives have been predicted to be potential candidates for spintronics,32–35 gas capture,36 and energy conversion.37–45 Using first principles calculations, Zhao et al.46 found that the 2D Cr–Pc monolayer exhibits high catalytic activity toward CO oxidation at room temperature. Wang et al.47 theoretically reported that FePc shows high activity for the ORR in an acidic solution due to the stable binding of Fe atoms with Pc monolayers and the coordinative unsaturated state of the doped active center, which has been confirmed by recent experimental work.48 All these previous studies suggest that the experimentally available TM@Pc monolayers with uniformly distributed single-atom active sites can be promising candidates for SACs.To date, a series of metal-doped phthalocyanine (MPc) monolayers, whose structure is similar to that of metal-doped porphyrins with one metal atom connecting to four nitrogen atoms, have been successfully synthesized in experiments with single metal atoms orderly and strongly anchored into the pores of the Pc.17,49,50 The advances in DFT have made it possible to accurately describe catalytic reactions,51 and the computational investigation on the OER/ORR performance of TM@Pc monolayers can shed some light on developing low-cost, effective and stable electrocatalysts, or on future improvements of the systems. Our calculations demonstrate that Rh@Pc monolayers exhibit efficient catalytic activity toward both the OER and ORR with stability both thermodynamically and kinetically.In this work, the catalytic performance of 2D transition metal doped Pc monolayers  as potential electrocatalysts toward the OER and ORR will be systematically screened with density functional theory calculations. This systematic study will be useful to help experimentalists to understand and select these transition metal elements, as these metals have exhibited OER and ORR activities in various other 2D materials.2.Ab initio Simulation Package (VASP) code.52,53 The projector augmented wave (PAW) pseudopotentials were used to describe the electron–ion interactions.54 The Perdew–Burke–Ernzerhof (PBE)55 functional of the generalized gradient approximation (GGA)56 was used to describe the electron–correction interactions. The van der Waals (vdW) interactions were described using the Grimme's DFT-D3 correction method.57 A plane-wave cutoff energy of 500 eV was adopted for all the computations to describe all atoms' valence electrons. Geometry optimizations were performed until the atomic force became less than 10−2 eV Å−1. The energy was converged to be less than 10−5 eV. A vacuum space of 20 Å was used to prevent the interaction between the periodic images. The Brillouin zone was sampled with 5 × 5 × 1 Monkhorst–Pack k-meshes.58 Bader charge analysis was performed to evaluate the charge transfer process.59 To investigate the stabilities of the screened out catalysts that possess excellent performance for the OER and ORR, the TM diffusion barriers were calculated by the climbing image nudged elastic band (CINEB) method.60,61Ab initio molecular dynamics (AIMD) calculations were also carried out to evaluate the corresponding dynamic stability. The algorithm of the Nose thermostat was used to calculate the canonical ensemble62 at 300 K for 10 ps with a time step of 2 fs. The implicit solvent model was used to simulate the solvent environment throughout the whole process using VASPsol under the water conditions.63 The details of OER and ORR calculations are provided in the ESI17 The adsorption Gibbs free energy is defined as the following Gadsorbent+catalyst, Gcatalyst, and Gadsorbent are the Gibbs free energy of the adsorbent on the catalyst, the isolated catalyst, and the isolated adsorbent, respectively.The spin-polarized density functional theory calculations were carried out with the Vienna 3.26,28,32,33,46 In the optimized TM@Pc monolayer structure, one TM atom binds with four inwardly projecting N atoms, forming four TM-N bonds. The binding energy (Eb) is defined as Eb = ETM@Pc − EPc − μTM, where ETM@Pc and EPc are the total energies of the TM@Pc system and the Pc substrate, respectively. μTM is the chemical potential of the TM atom calculated from its bulk crystal. Since μTM is referenced with respect to its bulk metal, negative values of Eb  has been used to analyze the interaction strength between the adsorbate and the catalyst.65–69 We have thus plotted εd as the center of mass position of the d-band partial density of states (PDOS) in Fig. S3.εd to a lower energy position with respect to the Fermi level is seen as the d-electron number of the TM atom increases at least when the TMs are in the same row of the periodic table. Because the interaction between the doped TM and intermediate species occurs due to the hybridization of their electronic states, the larger d-electron number of the TM atoms and their corresponding lower energy of εd generally result in weaker interaction strength with the adsorbates.70 Thus, the expected interaction strength between TM–Pc and intermediate species should have the following trend: Mn > Fe > Co > Ni > Cu, Ru > Rh > Pd and Ir > Pt. To verify the above assumption, we plot the Gibbs free energy value of intermediates  with various d-electron numbers of the TM@Pc systems in Fig. S4.εd as exhibited in Fig. S3.εd and Gibbs free energy of intermediates, at least when the TM atoms are in the same row of the periodic table. This phenomenon was also observed in previous experimental and theoretical studies.17,18,71,72 Accordingly, the interaction strength can be modulated to an optimal value by tuning the TM doped on the Pc sheet toward OER and ORR performance.The investigation of the distinct electronic properties of different types of TM atom doped Pc monolayers can help us better understand their catalytic activities. The d orbitals of the doped TM atoms on Pc monolayers were computed and are shown in Fig. S3.et al.,73 the adsorption Gibbs free energies of three intermediates  on the TM@Pc catalyst govern the intrinsic catalytic activity toward the OER and ORR. Three descriptors, Gibbs free energies of adsorbed HO*, O* and HOO*, are used to evaluate the catalytic activity of an OER/ORR electrocatalyst. According to the Sabatier principle,74 both too strong and too weak interaction strength between the intermediates and catalysts lead to adverse effects on the catalytic performance. Therefore, identifying promising electrocatalysts with moderate interaction of the reaction intermediates is one of our goals. As an ideal catalyst at the U = 0 V condition which can occur at its thermodynamic limit, it requires that the free energy barriers between two adjacent intermediate states for all the mentioned four electron transfer steps  is determined by the maximum difference between the two adjacent Gibbs free energies, while the overpotential of the ORR (ηORR) is determined by the minimum difference between the two adjacent Gibbs free energies. Obtaining the relationship among these three Gibbs free energies will simplify the analysis and the search for the optimal catalysis.75,76 Here, the scaling relationship of ΔGHO*vs. ΔGHOO* for all the considered TM@Pc catalysts is shown in GHOO* can be expressed as a function of ΔGHO*via equation ΔGHOO* = 0.82ΔGHO* + 3.14, with a high coefficient of determination (R2 = 0.992). The slope close to unity in the correlated adsorption free energies of HO* vs. HOO* reflects the fact that both HO* and HOO* have a single bond between the O atom and TM, which is similar to the cases of metal and metal oxide surfaces.77,78 Thus, the overpotential (ηOER and ηORR) as a function of four variables  can be reduced to two independent variables .73,78 As shown in the following: ΔGa = ΔGHO*, ΔGb = ΔGO* − ΔGHO*, ΔGc = ΔGHOO* − ΔGO* = (0.82ΔGHO* + 3.14) − ΔGO* and ΔGd = 4.92 − ΔGHOO* = 4.92 − (0.82ΔGHO* + 3.14). Thus, knowing only two descriptors, ΔGHO* and ΔGO* − ΔGHO*, is sufficient for us to describe the catalytic performance of a system toward the OER and ORR. In ηOER as a function of two independent descriptors ΔGHO* and ΔGO* − ΔGHO*. The blue region of the plot shows the highest activity area with ηOER reaching a minimum value of 0.21 V under the optimum condition (ΔGa = ΔGb = ΔGc = 1.44 eV). Note that the minimum value of the overpotential is not zero since the relationship ΔGHOO* = 0.82ΔGHO* + 3.14 excludes the ideal case of ΔGHO* = 1.23 eV and ΔGHOO* = 3.69 eV. Different catalysts fall on different points in the volcano plot of ηOER = 0.41 V) followed by Rh@Pc (ηOER = 0.44 V). The free energy diagrams of all the intermediate states of Rh@Pc and Ir@Pc toward the OER are shown in U = 0 V. Notably, the calculated ηOER values of Rh@Pc and Ir@Pc are comparable or even lower than those of the current best catalysts RuO2 (ηOER = 0.42 V)78 and IrO2 (ηOER = 0.52 V).77 Co@Pc also shows good activity (ηOER = 0.50 V) for the OER with the O* formation being the rate-determining step. The ORR is the reverse reaction of the OER. In η values for the ORR on various TM@Pc catalysts were compared. Under the optimal condition (−ΔGa = −ΔGd = 0.98 eV), the theoretical ηORR is found to be as low as 0.25 V. From the volcano plot in ηORR value of 0.44 V and the rate-determining step is the reduction of O2 to HOO* (ηORR = 0.55 V). This thermodynamic limiting overpotential is even lower than that of Pt (111) (ηORR = 0.48 V).79 Fe@Pc and Co@Pc also exhibit good catalytic activities (ηORR = 0.58 and 0.58 V) for the ORR with the rate-determining steps of reduction of O* to HO* and HOO* formation, respectively. Here, it should be noted that Rh@Pc can be used as the efficient bifunctional electrocatalyst for both the OER and ORR with an ηOER value of 0.44 V and an ηORR value of 0.44 V, respectively. To evaluate the dynamic stability of this promising catalyst, we have performed AIMD simulations under 300 K condition for 10 ps  to evaluate the stability of the doped metal centers against dissolution due to the proton attack of the active region using the equation:75 TM@Pc + nH+ → nH@Pc + TMn+. Where n refers to the oxidation state for the TM atom, nH@Pc refers to the Pc monolayer with the TM vacancy adsorbed by n number of hydrogen atoms ( + Gn+)(TM − G(TM@Pc) − nG+)( and G(TM@Pc) can be calculated directly, and ΔG+)(H = 0.5 × G2)(H − ln 10 × kT × pH = 0.5 × [E2)(H + ZPE2)(H − TS2)(H = 0.26 eV.17 As for Gn+)(TM = Gn+). Thus: Gn+)(TM = ΔGn+), where G is calculated with DFT, and ΔGn+)(TM is obtained from the literature80 and is listed in Table. S3.Gdiss(0), is calculated and shown in Gdiss(0) values are less than zero, which means they will be unstable against dissolution. However, as the pH value increases, ΔGdiss(pH) will also increase, thus, there will be a critical pH value, above which the TM@Pc catalyst will be stable. The critical value can be calculated by pHmin = −ΔGdiss(0)/(n × 0.0591) . It should be noted that both Rh@Pc and Ir@Pc can remain stable against dissolution under all pH conditions. This study highlights a potentially efficient new class of SACs based on the Pc monolayers toward the OER and ORR.To summarize, we have systematically screened a series of single TM atom doped Pcs as potentially efficient and stable SAC bifunctional electrocatalysts for both OER and ORR catalytic processes using a computational screening approach. Based on the computations of binding of TM atoms with Pc monolayers, we found that all the considered TM atoms exhibit a strong interaction with Pc monolayers as potentially stable SACs with high diffusion energy barriers. With the increase of the d-electron number of the doped TM atom on Pc monolayers that leads to the lower d-band center, the interaction strength between intermediates and the active doped TM atoms will decrease, which allows us to select the optimal TM@Pc catalyst toward the OER/ORR by tuning the doped TM element. According to the volcano plots of the OER and ORR, among all the studied TM@Pc catalysts, the best catalyst for the OER is Ir@Pc with an There are no conflicts to declare.NA-002-C9NA00648F-s001


We relied on Bland-Altman analysis to quantify the agreement between results recorded in the two positions. Using body surface charts, we tested the hypothesis that posture-induced variability stems from differences in exposed skin area.Eighty-two adults (42 men and 40 women), aged 26.1 ± 8.4 y (mean ± standard deviation), body mass index = 23.6 ± 4.8 kg/m2. When body volume was expressed in terms of the actual area of exposed skin in the compact position, the percent body fat bias became 0.08%, with a 95% confidence interval of %.Switching from compact to relaxed position resulted in a bias of -197 mL for body volume, -1.53% for percent body fat, and 1.085 kg for fat-free mass. The body surface area in contact with air was larger in relaxed position by 3632 ± 522 cm® should be controlled to avoid changes in the amount of air maintained under isothermal conditions by the body.Subject posture is a source of significant variability in air displacement plethysmography. The disagreement between results obtained in different positions can be eliminated by adjusting the surface area artifact, suggesting that subject positioning in the BOD POD As a ro2),2≤ BMI ≤ 39.9 kg/m2, and2).According to the modified Lund-Browder chart  S1 Fig)S1 Fig), 22 ≤ BMI ≤ 39.9 kg/m22.Hence, in the compact position, the corrected body volume was expressed as follows:BF was computed using Siri’s formula Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Overall, this is a really good study and I adds to the body composition literature on subject positioning using the BodPod. I encourage the author to correct past tense as well as grammar issues.Abstract:Line 29: I’d just state how many were in the study Eight-two healthy adults (42 men and 40 women etc….) How is healthy defined?Line: 31. How is “extreme” defined? I suggest just taking it out and saying “two different position were utilized.Introduction:Line 60: The accuracy of ADP was confirmed 61 for various categories of subjects, including healthy adults , elderly , and children . I would change was confirmed to The accuracy has been validated in various subjects……Line 108: Take out “has”Line115-116: Research doesn’t prove it merely suggests. Reword this sentence please.Methods:Line 135: What about nicotine or caffeine or alcohol consumption?Line 136: Why did the void before arrival? Usually you have subjects VOID right before testing.Line 200: Change “were done” to were analyzed.Discussion:Bringing the reader back to the purpose would be good.Change all ref to the actually author Jones-Smith et al..Reviewer #2: Thank you for your in-depth investigation of factors influencing calculation of the SAA for adults undergoing ADP assessments! See the attached file for this reviewer's comments/questions/suggestions.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentPLoS Comments to the Authors.docxSubmitted filename: Click here for additional data file. 17 Feb 2022Response to Reviewers We thank the reviewers for the thorough analysis of our manuscript! We equally appreciate their positive comments, which encourage us, and their constructive criticism, which hopefully helped us to improve the paper.  The reviewers’ comments are listed in this letter point-by-point, placed between quotation marks. Each comment if followed by our answer. In the revised manuscript, the modified portions of the text are highlighted using the Track Changes function of Microsoft Word. Line numbers are also given wherever the new text appears on a different line in the revised manuscript than in the original one. Reviewer #1 Comment 1: "Overall, this is a really good study and adds to the body composition literature on subject positioning using the BodPod. I encourage the author to correct past tense as well as grammar issues."We thank Reviewer #1 for the appreciative comment and for the help with pinpointing English usage issues. Comment 2: Line 29: "I’d just state how many were in the study Eight-two healthy adults (42 men and 40 women etc….) How is healthy defined?" We revised the Abstract accordingly. Comment 3: Line: 31. "How is “extreme” defined? I suggest just taking it out and saying “two different position were utilized." Done. Comment 4: Line 60: "The accuracy of ADP was confirmed 61 for various categories of subjects, including healthy adults , elderly , and children . I would change was confirmed to The accuracy has been validated in various subjects……" Done. Comment 5: Line 108: "Take out “has”" Done (line 109 in the revised manuscript).Comment 6: Line115-116: "Research doesn’t prove it merely suggests. Reword this sentence please." Done (lines 116-117 in the revised manuscript). Comment 7: Line 135: "What about nicotine or caffeine or alcohol consumption?" Indeed, we asked them not to consume alcohol for two days prior to being tested, but we did not mention smoking and coffee consumption. We revised the text accordingly (line 136 in the revised manuscript). Comment 8: Line 136: "Why did the void before arrival? Usually you have subjects VOID right before testing." Actually, we asked them to visit the bathroom right before commencing the first test. Hopefully, the rewritten sentence is less confusing than the original (line 138 in the revised manuscript). Comment 9: Line 200: "Change “were done” to were analyzed." Done (line 202 in the revised manuscript). Comment 10: "Change all ref to the actually author Jones-Smith et al.." Done. Reviewer #2 Comment 1: "Thank you for your in-depth investigation of factors influencing calculation of the SAA for adults undergoing ADP assessments! Your findings and insights provide great depth of understanding as to why body positioning within the BodPod chamber is critical – especially for serial assessments of body composition." We thank Reviewer #2 for this encouraging comment! Comment 2: "To conform to the conventions and grammar of the English language, this reviewer suggests enlisting services of a native English speaker to help clarify your otherwise well-written report. Suggestions for both grammar, clarity, and content are below. If the journal provided editing services, this list would be much shorter." We appreciate the constructive criticism and the proposed improvements of English grammar and style! Line 48 – "“compartment” should be “component”" Done. Line 67 – "by “softer” do you mean “less dense”? Please clarify" The revised sentence states explicitly that “softer” refers to “more compressible”. Line 70 - "change to read “as well as is a …”" Done. Line 72 - "change to read “hair be completely…”" Done (line 73 in the revised manuscript). Line 84 – "this reviewer did not see the definition of BSA and TGV prior to their use in the BV formula" The acronym of the body surface area (BSA) is defined in the revised text on line 71, whereas that of the thoracic gas volume (TGV) appears on line 77.Line 113 – "change to read “positions that concern”" The corresponding sentence was revised to state explicitly that the investigated positions differed in the surface area artifact, while being in accord with the manufacturer’s instructions. Line 128 – "use “lower and upper bounds” instead of “range” in table title" Done. Line 125 – "move “, including scale calibration,” to follow “procedure in line 134" Done (line 135 in the revised manuscript). Line 138 – "indicate if participants were barefoot for standing height" Done (line 140 in the revised manuscript). Line 141 – "change to read “wore dry, minimal….”" Done (line 142 in the revised manuscript). Line 153 – "be sure the sequence of posture in Fig 1 matches specifics of the figure title." Yes, the caption of Figure 1 is correct: panels A and B represent the subject in relaxed position, whereas panels C and D depict the subject in compact position, in which a significant part of her/his body surface area is hidden from the surrounding air. Line 154 – "would be helpful to identify if participants exited the chamber between trials in Tucker’s study."  Yes. they did exit the measurement chamber after every trial (line 161 in the revised manuscript).Line 158 – "even though you are most interested in SAA, indicated if TGV was estimated or measured? If estimated, mention that as a limitation." The TGV was predicted by the BOD POD’s software, as stated on line 144. Line 163 – "change to read “In the compact…”" Done (line 163 in the revised manuscript). Lines 185-189 – "is “VTG” supposed to represent “TGV”? The latter is a more common term, Be sure to define the term prior to its use."  We thank Reviewer #2 for noticing this typo! The correct acronym is defined before its first use (line 77). Line 193 – "insert a space before ±" Done. Line 255 – "references to SI figures no longer follows ascending numbering sequence. Reference to S Fig 3 occurs on line 170, but no reference to S figures 4 -6 found prior to reference to S7 figure. Reference to S44-S6 comes on line 274. See below for comments regarding clarity of the figure titles/legends." We chose to group the BA plots derived from the entire sample, and then, figures S7-S11 represent the BA analysis of data obtained for women, whereas figures S12-S15 refer to men. This option is explained in lines 252-253. Line 277 – "Is it not more appropriate to indicate that it is “The results from this study are in accord…”?" We revised the sentence accordingly (line 275 in the revised manuscript). Line 282 – "consider changing to read “positions that concern the fraction…”" Done (line 280 in the revised manuscript).Line 293 – "consider using “Peeters and colleagues [18]” instead of “ref [18]” as the subject of your sentence" The revised sentence is, hopefully, more straightforward and legible (line 291 in the revised manuscript). Line 303 – "change “ref [13]” to the first author’s name followed by [13]." Done (line 300 in the revised manuscript). Line 307 – "change to read “in close vicinity to the”" Done (line 304 in the revised manuscript). Line 313 – "consider clarifying who/what is being referred to aby “Both of them”. It seems you are referring to techniques, but it is not clear as written." The revised sentence opening avoids the pronoun: “Both the Lund and Browder chart and the “Rule of Nines” have been revisited ...” (line 309 in the revised manuscript).Line 316 – "by “This study” are you referring to “The present study” meaning your study? Not clear as written." Indeed, it is clearer to write “The present study” (line 312 in the revised manuscript). Comment 3: "This reviewer did not see a reference to S13 or S16." Supplementary figures S7-S11 show the BA plots obtained for women, whereas S12-S15 refer to men. These sex-specific plots are mentioned on line 253 and the corresponding BA parameters are listed in Table 3. Comment 4: "In reference list, check format for journal title for #19. It seems to be the only one that spells out the complete journal title." The revised reference contains the abbreviated journal title (Int J Exerc Sci). Comment 5: "My primary concern is with the figure titles/descriptions of the Bland and Altman SI plots using both red and black line colors. Some of the SI figures have two solid red lines with 95% LOA dashed lines, but no indication I found as to what they were indicating. While this suggestion may be perceived by the authors as adding redundancy, it would clarify some confusion for readers like me who have extensive experience with Bland & Altman plots. It would also help with the concept about each figure being able to stand alone." The revised caption of S4 Fig. provides a detailed presentation of the notations and the elements displayed on each BA plot (lines 473-478). Furthermore, to guide a reader interested in another supplementary figure, the captions of S5-S15 Figs. were supplemented with the sentence “Notations are explained in the caption of S4 Fig.”. Comment 6: "Another concern was the inability to understand “the numbers displayed …” aspect of the title for SI Fig. Providing an example would go a long way in providing clarity of what you are trying to say." The explanations given in the caption of S4 Fig. address this problem, too.AttachmentADP_posture_Response_to_reviewers.docxSubmitted filename: Click here for additional data file. 4 Apr 2022The impact of subject positioning on body composition assessments by air displacement plethysmography evaluated in a heterogeneous samplePONE-D-21-15286R1Dear Dr. Neagu,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Carla PegoraroStaff EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: Very fun to read again! Nice job on addressing grammatical issues and providing additional clarity. Please check line 243 and change "SSA" to "SAA" if it is indeed a typo. Good job, and congratulations!**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: No 7 Apr 2022PONE-D-21-15286R1 The impact of subject positioning on body composition assessments by air displacement plethysmography evaluated in a heterogeneous sample Dear Dr. Neagu:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. 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Combining the optimized carrier concentration by Cl doping and enlarged Seebeck coefficient through activating multiple conduction bands evolutions with temperature, favorable power factors are maintained. Besides, the electron doping stabilizes the phase of PbSnS2 and the complex-crystal-structure induced strong anharmonicity results in ultralow lattice thermal conductivity. Moreover, a maximum power generation efficiency of ~2.7% can be acquired in a single-leg device. Our study develops a n-type sulfide PbSnS2 with high performance, which is a potential candidate to match the excellent p-type SnS.Great progress has been achieved in p-type SnS thermoelectric compound recently, while the stagnation of the n-type counterpart hinders the construction of thermoelectric devices. Herein, n-type sulfide PbSnS 2 crystals enabled by multiple bands convergence.Direct and reversible conversion between heat and electricity can be achieved in thermoelectric materials. Here, the authors realize high thermoelectric performance in PbSnS The efficiency of thermoelectric devices depends on the material’s dimensionless figure of merit, ZT = S2σT/(κlat + κele), where S, σ, S2σ, T, κlat and κele represent Seebeck coefficient, electrical conductivity, power factor, absolute temperature, lattice thermal conductivity and electronic thermal conductivity, respectively. Numerous strategies have emerged to boost thermoelectric performance after decades of efforts, including enhancing Seebeck coefficient through distortion of the density of states4, band convergence7 and electron energy barrier filtering8, optimizing carrier concentration and maintaining high carrier mobility to improve electrical conductivity12, and designing all-scale hierarchical architectures to lower lattice thermal conductivity15. The potential of thermoelectric materials can be further motivated through applying these classical strategies synergistically.The thorny problems of energy shortage and environmental pollution have been challenging the sustainable development. Thus, thermoelectric materials have gained increasing attention because they provide an alternative way of energy utilization by realizing the direct and reversible conversion between heat and electricityZT of 1.25 was reported in SnS0.91Se0.09 crystals16, mainly due to the synergistically optimization of carrier effective mass and mobility through dynamic valence bands interplay. However, the development of n-type SnS is far lagging behind due to the great difficulties and challenges on achieving effective electron doping to optimize the poor n-type electrical transports.The ideal thermoelectric devices for large-scale and high-efficiency applications prefer homojunction structures in order to avoid lattice mismatch incompatibility and harmful band misalignment. Therefore, the development of similar-system and performance matched n-type and p-type thermoelectric materials is particularly important. Recently, the low cost and environmentally friendly p-type SnS has been greatly developed and a high average 19, deviating from stoichiometric ratio to compensate for Sn vacancies20, and aliovalent anion doping (Cl and Br)24. However, the electron carrier concentration obtained through the above approaches is far below the optimal carrier concentration level required for high-performance thermoelectric materials25. The remaining one glimmer of hope is that isovalent cation Pb alloying favors electron generation through suppressing Sn vacancies and forming interstitials Sni and Pbi27. Meanwhile, researchers have concluded that the solid solubility of Pb element in SnS is slightly more than 50%29, and the solid solubility of Pb in Sn positions will significantly favor the effective electron doping in SnS26. By using Pb alloy at the Sn sites in SnS, another promising sulfide compound, PbSnS2, was obtained to approach promising n-type thermoelectric performance. Surprisingly, similar to SnS and SnSe33, PbSnS2 is composed of low-cost elements, possesses the layered and orthorhombic crystal structure with Pnma space group, the wide bandgap ~0.9–1.12 eV, and the intrinsically low thermal conductivity35. All of these completely meet the selection rules of potentially high-performance thermoelectric materials36. However, apart from the theoretical calculations37, PbSnS2-based thermoelectrics have few experimental achievements due to its ultralow carrier concentration and poor electrical conductivity26, making it rather urgent to develop and establish the proper n-type transports for PbSnS2 as the counterpart of p-type SnS.To date, several attempts to realize n-type SnS have been conducted, including aliovalent cation doping 40. Herein, we successfully synthesized PbSnS2 crystals by a modified temperature gradient method and investigated their thermoelectric transport performance in detail. Compared with the in-plane direction, our results show that higher thermoelectric performance is achieved along the out-of-plane direction due to the ultralow thermal conductivity coming from strong interlayer phonon scattering and superior power factor at higher temperatures (>600 K) due to increased interlayer charge density, which is similar to the anisotropy of n-type SnSe38. The undoped PbSnS2 crystal shows typical semiconductor transport behavior with rising temperature due to the low carrier concentration. And it might be partially decomposed into PbS and SnS at ~623 K, while PbS as a high thermal conductivity phase is harmful to thermoelectric transport41. To improve the poor electrical transport performance of the pristine PbSnS2 crystals, the halogen element Cl was selected as electron dopant due to its similar ionic radius to S. Surprisingly, Cl doping enhances the phase stability of PbSnS2 crystals compared with the undoped sample, and their lattice thermal conductivity follows the Umklapp mechanism to remain the low values42. The aliovalent substitution of Cl at S sites significantly optimizes the carrier concentration and thus increases the electrical conductivity. Also, the effective doping pushes Fermi level deeper and activates multiple conduction bands with increasing temperature, maintaining the large Seebeck coefficient. Resultantly, the power factor, lattice thermal conductivity and ZT values of the optimal Cl doped PbSnS2 crystal are ~4.26 μW cm−1 K−2, ~0.25 W m−1 K−1 and ~1.2 at 773 K, respectively, showing tremendous optimization after Cl doping. Additionally, certain performance of the output power and energy conversion efficiency is acquired in a single-leg device based on our high-performance PbSnS2 crystals. This study demonstrates that Cl doped PbSnS2 crystals can be the promising n-type thermoelectric candidates. Our attempts on single-leg devices also develop a potential n-type counterpart of p-type SnS to form low-cost and full-scale thermoelectric devices.Strategies of growing crystals and utilizing the unique layered crystal structure have been developed to optimize the electrical performance in both SnSe and SnS crystals2, X-ray diffraction (XRD) analysis and scanning transmission electron microscope (STEM) characterization were performed. A typical PbSnS2 crystal prepared by the modified temperature gradient method and the measurement samples along different crystalline directions are shown in Fig. 2 (carrier concentration is 3.6 × 1011 cm−3) and Cl doped PbSnS2  prove that the single phases were synthesized , and its electrical transport performance cannot be estimated accurately within the detection ability of the instrument 48, as shown in Fig. σ = nHeμH (where e is electron charge), the Hall carrier mobility decreases following the law of ~T −1.5 with rising temperature, demonstrating that the carriers are mainly scattered by acoustic phonons49. On the contrary, according to the temperature-dependent electrical conductivity and Seebeck coefficient, the weighted mobility decreases following the law of ~T −1.5 at a relatively lower temperature range (~300–473 K), while completely deviates from this law at higher temperatures (~473–823 K) for Cl doped crystals. Namely, the calculated weighted mobility is much higher than the theoretical T −1.5 line. It is well known that the weighted mobility is related to the Hall carrier mobility by μW ≈ (m*/me)1.5μH (where me is the electron mass)47. Thus, density of states effective mass m* is effectively improved at high temperatures, which is contributed from the multiple band transport as discussed later. Combined with the promoted carrier concentration and electrical conductivity by Cl doping and favorable Seebeck coefficient caused by larger effective mass at high temperatures, the power factor of PbSnS2 crystal maintain a relatively high value over the whole temperature range  and the conduction band minimum (CBM) of the optimal Cl doped sample are located at Y point and Γ-X direction, respectively. Therefore, PbSnS2 is an indirect band gap semiconductor with a band-gap of ~0.9 eV, which is in good agreement with previous calculations44 and our experimental values  can be activated by electron doping at room temperature. We further investigate the evolution of conduction band with the temperature. Namely, the electronic band structures at 300, 573, 623, 673 and 773 K are aligned to the CBM1  data of the optimal Cl doped sample , heat capacity (Cp), Lorenz number (L), and electronic thermal conductivity (κele), and sample density (ρ) were presented in Supplementary Fig. 2 polycrystal35 and crystal maintain ultralow values over the whole temperature range comparing with other n-type lead and tin chalcogenides, including Cl doped PbS polycrystal45, Br doped SnS crystal23, and Br or Cl doped SnSe crystals38  were calculated based on DFT calculations. Figure 2 along Γ-X, Γ-Y and Γ-Z directions are the softest among the typical lead and tin chalcogenides like PbS, SnS and SnSe  around the Sn and Pb atoms (the isosurface level is set as 0.95), which is an obvious sign of the presence of lone pair electrons  value of ~0.3 and ~1.2 can be achieved in the optimal Cl doped crystal at 300 and 773 K, respectively, along with a high average ZT (ZTave) value of ~0.75 within 300–773 K . Furthermore, it is apparent that the thermoelectric transport performance of Cl doped PbSnS2 outperforms that of Br-doped SnS crystals over the whole temperature range due to the much higher carrier concentration in PbSnS2 crystals24. The high thermoelectric transport performance of n-type PbSnS2 crystal indicate that it is expected to be a counterpart of p-type SnS crystal to fabricate low-cost and highly efficient thermoelectric devices16.Combined with the tremendously improved electrical transport performance and intrinsic low lattice thermal conductivity in Cl doped PbSnSion Fig. . It can 2 crystals experimentally, the single-leg power generation test was performed using mini-PEM equipment, as shown in the inset of Fig. Tc = 295 K and Th = 672 K  were weighted according to the nominal compositions of PbSnSx2-Clx  and were flame-sealed into quartz tubes after vacuum. In order to prevent the sample from oxidizing due to quartz tubes rupture during the phase transformation, outer glass quartz tubes were used. These prepared samples were slowly heated up to 873 K for 10 h and then soaked at this temperature for 12 h, next heated up to 1223 K for 10 h and then soaked at this temperature for 6 h in muffle furnace. Subsequently, then polycrystalline ingots would be obtained after cooling in the furnace. The obtained ingots of ~15 g were ground into powder and put into special quartz tubes with taper. After being vacuumized and flame-sealed, the reprocessed ingots were heated up to 1313 K for 12 h and then soaked at this temperature for 10 h, next slowly cooled to 973 K at a rate of 1 K h−1 in a vertical furnace with a temperature gradient. Subsequently, PbSnS2 crystals with size of ~Φ11 × 40 mm3 were obtained after cooling in the furnace.High-purity raw materials Pb block , Sn particles , S powder , and PbCl3 along the in-plane and out-of-plane directions. The Seebeck coefficient and conductivity of the samples could be obtained with Ulvac Riko ZEM-3 instrument simultaneously in a thin helium atmosphere. It should be noted that the sample surface is coated with a thin layer of boron nitride to prevent contamination of the instrument by possible volatilization, and that the uncertainty of Seebeck coefficient and conductivity measurements is within 5%.The obtained crystals were cut and polished into rectangles with size of ~ 3 × 3 × 7 mmΦ6 × 1.5 mm3 along the same direction as the electrical transport performance measurements. The samples were coated with a thin layer of graphite to reduce the error caused by the emissivity of the material. The total thermal conductivity can be calculated via κ = DCpρ, where D is the thermal diffusion coefficient, which could be obtained using Netzsch LFA 457 and analyzed using the Cowan model with pulse correction. Cp represents specific heat capacity, which can be calculated according to Debye model and we have compared the Cp values near the temperature region of the structural phase transition based on Dulong-Petit law, Debye model and the experimental DSC data. ρ represents the density, which can be inferred from the dimensions and mass of the samples. The uncertainty of the total thermal conductivity is within 15% due to errors in measurement or calculation of D, Cp, and ρ. Therefore, the uncertainty of ZT values obtained by the final calculation is within 20%.The obtained crystals were cut and polished into slices with size of ~Scanning transmission electron microscopy (STEM) studies were conducted using a Cs-corrector JEM-ARM200F NEOARM with a cold FEG at 200 kV. The thin specimens were prepared by conventional standard methods, that is, cutting, grinding, dimpling, polishing and Ar-ion milling on a liquid nitrogen cooling stage.2 and the optimal Cl doped PbSnS2 crystal were ground into powder in an N2-filled glove box, and then passed through a 400-mesh sieve before being loaded into cylindrical quartz capillary tubes with a diameter of ~0.5 mm and flame-sealed. The temperature-dependent phase structures of the samples were obtained at BL14B1 of Shanghai Synchrotron Radiation Facility (SSRF) using X-ray with a wavelength of 0.6887 Å. The samples were heated from 300 K to 823 K at a rate of 10 K/min during the measurement and GSAS software was adopted to refine the phase structures with various temperature.Samples of undoped PbSnS2 crystal with the geometrical dimension of ~2 mm (length) × 3 mm (width) × 6 mm (height), where nickel was electroplated onto the cleavage surface as a barrier layer and gold foil was used as contact material. Mini-PEM Ulvac-Riko (Japan)59 was adopted for the direct data of output power and conversion efficiency while the cold-side temperature (Tc) was maintained at 295 K and the hot-side temperature (Th) was varied from 397 K to 672 K.The single-leg device was fabricated using the optimal Cl doped PbSnSSupplementary InformationPeer Review File


Objectives: To investigate and evaluate the key factors related to job satisfaction performance of home healthcare nurses (HHNs).Methods: A total of 31 HHNs from three community hospitals in Zhejiang province were invited to participate in the study. They completed a questionnaire survey based on the home healthcare nurse job satisfaction scale (HHNJS) from February to March 2022. Consistent fuzzy preference relation (CFPR) methods and important-performance analysis (IPA) were used to obtain the attribute weights and performance for HHNs job satisfaction.Results: The results showed that the attributes of C13, C14, C15, C23, C24, C42, C51, and C52 were key factors influencing HHNs job satisfaction.Conclusion: The hybrid multiple-criteria decision-making (MCDM) model can help home-healthcare-agency administrators better understand the key factors related to HHNs job satisfaction and establish reasonable improvement strategies. The coronavirus disease COVID-19) is a highly communicable disease that has led to more than 257 million infections and 5000 thousand deaths worldwide 9 is a hi. During In addition to their standard care for patients without COVID-19 during the pandemic, nurses have worked hard to care for sick patients, provide comfort in the face of death, and educate the public about protection measures to stop the SARS-CoV-2 spread . Many nuThe Home Healthcare Nurse Job Satisfaction Scale (HHNJS) is a reliable and valid instrument for measuring the job satisfaction of HHNs. It includes eight subscales—four focus on relationships and four on other work aspects . Some stOur study established a multiple-criteria decision-making (MCDM) model and applied it to the HHNs job satisfaction analysis. Consistent fuzzy preference relation (CFPR) was initially used to obtain HHNJS attribute weights before the importance-performance analysis (IPA) method was applied to analyze the job satisfaction of HHNs performance.Methods section introduces the HHNJS survey questionnaire and illustrates the CFPRs calculation procedures and IPA methods, Results section outlines the results of applying the hybrid MCDM model to the HHNs job satisfaction assessment, Discussion section discusses the results and limitations of the study, and Conclusion section presents our conclusions.This study is structured as follows: This study aims to develop an MCDM model and apply it to the community hospitals in the present study. The HHNJS instrument is an evaluation tool that includes eight subscales. The dimensions and criteria weights were then constructed using the CFPRs method. This model was ultimately used to evaluate and improve the satisfaction of HHNs in the community hospitals in the present study. The study flow chart is shown in The HHNJS is the only scale specifically designed to measure HHNs job satisfaction . It was C1),” “relationship with peer (C2),” “professional pride (C3),” “salary and benefit (C4),” “relationship with physician (C5),” “relationship with organization (C6),” “autonomy and control (C7)” and “stress and workload (C8)” [The HHNJS comprises 30 items to measure the level of job satisfaction in eight components: “relationship with patients (d (C8)” . Each ofk attributes in the evaluation system, it needs to be compared AHP is a commonly used MCDM method to evaluate the relative attribute weights by performing pairwise comparisons . The decn effect , 29. Henn effect . This apn system . The rem results . CFPRs a results . Thus, t results , supplie results , logisti results , and man results , 31, 36.The IPA method was first proposed by Martilla et al. in 1977 in marketing  and has In quadrant I—keep up the good work—the attributes are both highly important and high performance. This means that these attributes are organizational strengths, and decision-makers should maintain resource input. In other words, nursing department decision-makers should maintain these attributes to maintain acceptable levels of HHNs job satisfaction.Attributes in the “possible overkill” quadrant have high customer satisfaction with performance but are unimportant. Resources invested in these attributes can be reduced and should be applied elsewhere. In the short term, nursing decision-makers can temporarily reduce their attention to these attributes and use related resources for Quadrant IV attributes to improve job satisfaction.Attributes in the “low priority” quadrant have low importance, poor performance, and can be temporarily ignored. Compared with other attributes, nursing department decision-makers can directly identify the resources related to these and other attributes that can improve job satisfaction.Attributes located in “concentrate here” are highly important but have poor performance, which indicates that the attributes require immediate improvement, and decision-makers should focus on them. Nursing decision-makers should increase their satisfaction with these attributes and divert resources from Quadrants II or III to these attributes.Thirty-one HHNs from three community hospitals in Zhejiang Province participated in the study and completed a questionnaire survey from February to March 2022. The questionnaire was comprised of three parts: the first evaluated the relative importance of the attributes, the second was a self-evaluation of job satisfaction with home healthcare, and the third covered participant demographics. 100% of participants were female, and 48% had a bachelor’s degree. 58% percent of nurses were younger than 30 years old, and most were senior nurses. 61% of participants had been nursing for less than 10 years, while 81% had worked in home healthcare nursing for no more than 5 years. C1)” dimension was the most important, with an average weight level of 0.1731, followed by “Salary and benefit (C4)” and “Relationship with peer (C2)”. Relative to the other dimensions, “Relationship with organization (C6)” was the least important. “I am treated as a professional colleague by physicians (C52)” was the most important criterion, with an average global weight of 0.0662, while “At times I am overwhelmed by all the work I have to do (C81)” was the least important, with an average global weight of 0.0134.Overall, the “Relationship with patients (The IPA results of HHNs job satisfaction are summarized in C13),” “My work is important and worthwhile (C14),” “The patient care that I provide adheres to my professional standards (C15),” “There is a good amount of collegiality among the nurses I work with (C23),” “I have peers whom I can rely on and turn to if necessary (C24),” “An upgrading of the pay scales at this agency is needed (C42),” “Physicians value my input on the status of their home healthcare patients (C51)”, and “I am treated as a professional colleague by physicians (C52).”Quadrant I includes “I have helped patients maintain or improve their quality of life (C11),” “The relationships that I have established with patients are rewarding (C12),” “The support I have from my nursing peers is a positive aspect of my job (C21),” “I can communicate comfortably with the nurses I work with (C22),” “If I had to do it over again, I would choose home healthcare as my area of practice (C31),” and “I am able to adjust the hours of my work if needed (C71).”Quadrant II includes “Patients are satisfied with the care that I provide (C41),” “I am satisfied with the professional relationship that I have with nursing administration at this agency (C61),” “I have more flexibility in my hours of work than nurses in other practice settings (C72),” “At times I am overwhelmed by all the work I have to do (C81),” “I am able to meet the demands of my job (C83),” “I am able to cope with the increased demands for documentation in home care (C84),” and “Sometimes I get frustrated because all my activities are programmed for me (C85).”Quadrant III includes “My present salary is satisfactory (C32),” “I am proud to talk to people about the work I do (C33),” “Nursing salaries at other agencies are better than salaries at this agency (C43),” “The benefit package at this agency is satisfactory to me (C44),” “I have the power to generate change in organizational policy at this agency (C62),” “I have the opportunity to grow and develop as a professional nurse within this agency (C63),” “I have sufficient control over scheduling my time (C73),” and “I have the independence to make important decisions in my day-to-day work (C74).”Quadrant IV includes “I would commend my job to another health care professional  have higher relative importance and priority than those in quadrant III. This indicates that home-healthcare-agency administrators should focus primarily on these attributes as they are the critical factors influencing HHNs job satisfaction.HHNs provide skilled patient care and spend most of their time interacting with them. A relationship is established when nurses care for patients , and dir32, C33, C43, C44, C62, C63, C13, C73, and C74) take precedence in improving job satisfaction performance in home healthcare nursing. Professional pride is key to nursing job satisfaction due to the positive image of nursing and pride in nursing skills. Threatening professional pride may lead to nurses choosing not to continue with nursing and not recommending the field to others [C73 and C74 is poor [Compared to other attributes, the attributes in quadrant IV  and IV . Combined with the attribute weights, home-healthcare-agency administrators should focus their attention and resources on the attributes in quadrant IV.The main findings of the study are as follows. First, we obtained the attribute weights of HHNJS and found that This study has a few limitations. First, the study used the optimized CFPR method based on the AHP method to explore the relative weights of attributes and identify key factors. Although CFPRs can overcome the shortcomings of AHP and improve its practical value effectively, they are still based on the independent relationship between attributes. Second, this study was a cross-sectional survey, limited by its self-report nature, and might not reflect the long-term HHNs job satisfaction performance. Finally, the study only included 31 HHNs in one prefecture-level city in China; therefore, the findings cannot be widely generalized. Future research using random selection over a wider group of nurses from different regions is recommended.This study used the hybrid MCDM model to investigate the critical factors and performance of HHNs job satisfaction based on nurse experiences in three community hospital. The findings can help home-healthcare-agency administrators better understand the critical factors influencing the HHNs job satisfaction and the current job satisfaction performance. This could enable them to establish reasonable strategies to improve job satisfaction and retain the nursing workforce.


Microcurrent is a non-invasive and safe electrotherapy applied through a series of sub-sensory electrical currents (less than 1 mA), which are of a similar magnitude to the currents generated endogenously by the human body. This review focuses on examining the physiological mechanisms mediating the effects of microcurrent when combined with different exercise modalities (e.g. endurance and strength) in healthy physically active individuals. The reviewed literature suggests the following candidate mechanisms could be involved in enhancing the effects of exercise when combined with microcurrent: (i) increased adenosine triphosphate resynthesis, (ii) maintenance of intercellular calcium homeostasis that in turn optimises exercise-induced structural and morphological adaptations, (iii) eliciting a hormone-like effect, which increases catecholamine secretion that in turn enhances exercise-induced lipolysis and (iv) enhanced muscle protein synthesis. In healthy individuals, despite a lack of standardisation on how microcurrent is combined with exercise (e.g. whether the microcurrent is pulsed or continuous), there is evidence concerning its effects in promoting body fat reduction, skeletal muscle remodelling and growth as well as attenuating delayed-onset muscle soreness. The greatest hindrance to understanding the combined effects of microcurrent and exercise is the variability of the implemented protocols, which adds further challenges to identifying the mechanisms, optimal patterns of current(s) and methodology of application. Future studies should standardise microcurrent protocols by accurately describing the used current  for specific exercise outcomes, e.g. strength and power, endurance, and gaining muscle mass or reducing body fat. Electrical currents of this magnitude are insufficient to excite motor nerves , the shape of the pulse, the direction the pulse flows in, the duration (time) of the stimulation. Some waveforms are exceedingly complex, with variations in amplitude, frequency, pulse-width, and polarity all occurring within a treatment cycle. Unfortunately, many investigations do not properly describe the characteristics of the applied current, making it difficult to elucidate what is the optimal current configuration to be applied in different circumstances and for different outcomes, e.g. maximise exercise adaptation (such as hypertrophy or body fat reduction), accelerate recovery or attenuate exercise-induced muscle damage. Furthermore, the electrode placement could also be a potential source of confusion. Nonetheless, the treatment pattern of some microcurrent devices such as the Arc4Health used by Naclerio et al.  and NaclTo the best of our knowledge, no study has examined the impact of current characteristics and the effectiveness associated with the expected outcomes. The following sections review the characteristics of the used microcurrent protocols, the associated outcomes, and the probable mechanism of action.Microcurrent therapy has been used in a clinical setting for many years, and its origin dates back a few centuries ago when electrostatically charged leaf was used to treat skin conditions such as skin lesions and skin ulcers Belanger . Some ofAs described in Fig. +). This is described by Mitchell’s chemiosmotic theory  receive a total of two electrons and decompose into a molecule of hydrogen (H2) and hydroxyl ion (OH−). This is the cathodic hydrolysis processes of reduction: 2H2O + 2e− → H2 + OH−. In the second process, two H2O molecules give the positive electrode (anode) a total of four electrons and decompose into a molecule of oxygen and four hydrogen ions (H+). This is the anode hydrolysis processes, oxidation: 2H2O → O2 + 4H+ + 4e−. Protons are formed on the anodic side, and they move from anode (+) to cathode (−). This flow of positive hydrogen ions towards the negative electrode is proposed as a mechanism of increased ATP production in the mitochondria. Molecules of ATP are formed through the H + -ATPase when they reach the mitochondrial membrane  using transcutaneously applied MCT was ~ 12–20%, 12–12% and ~ 10–13%, respectively. Conversely, smaller reductions of both visceral (~ 7%) and abdominal (~ 4%) fat were observed in the group performing only exercise (n = 9). Similarly, our research group analysed the effect of using MCT for 3 h post-workout or during the morning on non-training days for 8 weeks in 18 cross-country male athletes , the athletes allocated to the MCT group (n = 9) significantly (p < 0.05) decreased lower limb fat and showed a trend (p > 0.05 and < 0.1) to reduce whole-body fat  when using MCT during or shortly after the completion of a 30 min endurance exercise vs performing the exercise alone  male and female university students aged 18–30 years. Participants received a trans-abdominal microcurrent stimulation  or placebo  for 40 min prior to performing a single moderate aerobic exercise session. The MCT protocol had an intensity between 700 and 999 μA with a frequency of 10–25 Hz. Although both groups (MCT and placebo) showed significant increases (p < 0.01) and large effect sizes (d = 2.75 and 2.5 respectively) in the exercise-induced serum glycerol levels that were used as lipolysis marker indicating triglycerides hydrolysis (Lafontan and Langin p = 0.03). However, more recently, compared to a sham treatment (n = 20), no lipolytic effect was observed in response to a 40 min application of MCT (n = 18) in university students , comparing the effectiveness of different MCT protocols administered with different currents and frequencies are needed to establish the lipolytic effect of MCT, as well as the optimal time of application . Moreover, in order to explore the lipolytic effect of MCT, either delivered alone or combined with exercise, investigations using individuals with different adiposity levels (i.e. overweight and obese participants) are needed.Noites et al.  investigThe chronic effect of MCT to increase the exercise-induced lipolytic activity in healthy individuals  and long applications times of up to 3 h.n = 41), adding MCT (n = 42) to a 60 min aerobic session acutely increased lipolysis in young (18–30 years old) men and women. The microcurrent protocol was set at an intensity between 700 and 999 μA with frequencies of 10–25 Hz  significantly decreased subcutaneous fat measured by ultrasonography in young females participants (18–30 years old), compared to performing exercise alone  and high (1 to 999 μA) currents set at various frequencies. Nevertheless, protocols with lower frequencies (10–25 Hz) seem more effective in maximising exercise-induced lipolysis.p = 0.014) and muscular efficiency estimated by a markedly reduced root mean square values of the electromyography signals, along with an increased number of plantar flexions  performed in a single leg heel-rise test was reported in elderly individuals (n = 19) after receiving a 40 min MCT with an intensity of 25 μA and a frequency of 8 Hz  after receiving a 30-min frequency-specific microcurrent post-exercise with an intensity of 200 μA and frequency of 0.3–3.5 Hz  was observed for vertical jump and strength performance, only the MCT group significantly improved vertical jump height, while both groups, MCT and sham (only training treatment) similarly improved bench press strength. It was suggested that differences in the training routine configuration, involving a higher volume of work on the lower body than on the upper body musculature could have caused the observed difference  increased ATP resynthesis, (ii) maintenance of intracellular calcium homeostasis, (iii) enhanced exercise-induced lipolysis and (iv) promoted muscle protein synthesis.Within an exercise training context, MCT can accelerate post-exercise recovery, attenuate the severity of delayed-onset muscle soreness, and optimise physical training outcomes, e.g. skeletal muscle hypertrophic response, strength gains, endurance capacity and fat mass reduction.The available literature suggests that the abovementioned effects can be achieved when MCT protocols are applied incorporating currents set between 20 and 400 μA, in addition to longer application times (> 30 min up to 3 h). Nonetheless, the variability of the analysed MCT protocols adds further challenges in identifying optimal protocols in terms of the pattern of current(s), time of application and treatment duration associated with the expected specific clinical or exercise-related outcomes.From a practical perspective, MCT can be applied for 30–40 min prior to and after endurance exercises sessions to acutely stimulate lipolysis and accelerate tissue recovery. MCT can be regularly implemented during 3 h post-workout and at some point (e.g. morning time) on non-training days to maximise training-induced adaptations and attenuate DOMS after performing unfamiliar hard physical exercises. Even though no conclusive recommendation can be made regarding the optimal current characteristics, clinicians and coaches should consider current intensities set between 20 to 400 μA to attenuate DOMS or 20–999 μA with low frequencies (10–25 Hz) to maximise lipolysis and fat reduction.(i)Well-designed interventions, including participants of different ages (including older adults), sport disciplines, and level of performance should be designed to explore the optimal effective protocols associated with differentiated exercise-related outcomes, such as gaining muscle mass, reducing body fat, and improving recovery, strength, endurance, or power performance, and to attenuate the age-related decline in muscle mass and improving wellbeing.(ii)In order to identify the real impact of using MCT alone or combined with exercise, researchers are encouraged to conduct a thorough control of the training load configuration in terms of intensity, rest periods, volume, training frequency, duration and exercise modalities.(iii)The impact of some common uncontrolled variables, such as sleeping time and quality, mental wellbeing, diet patterns, and nutritional habits, should also be considered. For instance, combined approaches including the application of MCT alongside the ingestion of nutritional supplements such as high-quality protein or β-hydroxy-β- methylbutyrate (HMB), which is nitrogen-free and recommended for the elderly to counteract sarcopenia (He et al., Based on the current reviewed literature, the following considerations are proposed for futures studies: