Even-chain carboxylates (n-butyrate) were produced when using enantiopure and racemic lactate with lactate conversion rates increased in nZVI presence (1 g⋅L–1). Consumption of hydrogen and carbon dioxide was observed late in the incubations and correlated with acetate formation or substrate conversion to elongated products in the presence of nZVI. Lactate racemization was observed during chain elongation while isomerization to D-lactate was detected during propionate formation. Clostridium luticellarii, Caproiciproducens, and Ruminococcaceae related species were associated with n-valerate and n-caproate production while propionate was likely produced through the acrylate pathway by Clostridium novyi. The enrichment of different potential n-butyrate producers  was affected by nZVI presence and concentrations. Possible theories and mechanisms underlying the effects of nZVI on substrate conversion and microbiome composition are discussed. An outlook is provided to integrate (bio)electrochemical systems to recycle (n)ZVI and provide an alternative reducing power agent as durable control method.Medium-chain carboxylates (MCC) derived from biomass biorefining are attractive biochemicals to uncouple the production of a wide array of products from the use of non-renewable sources. Biological conversion of biomass-derived lactate during secondary fermentation can be steered to produce a variety of MCC through chain elongation. We explored the effects of zero-valent iron nanoparticles (nZVI) and lactate enantiomers on substrate consumption, product formation and microbiome composition in batch lactate-based chain elongation. In abiotic tests, nZVI supported chemical hydrolysis of lactate oligomers present in concentrated lactic acid. In fermentation experiments, nZVI created favorable conditions for either chain-elongating or propionate-producing microbiomes in a dose-dependent manner. Improved lactate conversion rates and n-caproate production were promoted at 0.5–2 g nZVI⋅L The metduce NAD . Then, pe (PFOR) . This twe (PFOR) . In the elsdenii  or propielsdenii .3– and SO42– present in the medium . Acetate can then be used by chain-elongating bacteria to produce MCC. However, CO2 recapture would be limited to 33–50% since homoacetogenic bacteria requires four H2 molecules to reduce two CO2 to acetate (H2/CO2 ratio = 2) while the stoichiometric H2/CO2 ratio produced from lactate is 1 or 2/3 when n-butyrate or n-caproate are produced, respectively (2 (experiment III) concentrations. Finally, chain elongation of enantiopure lactate (D-lactate and L-lactate separately) or a racemic mixture of both enantiomers was studied with and without nZVI (experiment IV).Pyruvate decarboxylation by PFOR is a carbon-diverging step in lactate-based chain elongation as it results in carbon lost as COoxylates , this coectively . To achiectively  or nZVI ectively . Lastly,ectively . Thus, aectively . Howeverectively . Abiotic–1; MnSO4⋅H2O, 1.0 g⋅L–1; Fe(SO4)2 (NH4)2⋅6H2O, 0.8 g⋅L–1; CoCl2⋅6H2O 0.2 g⋅L–1; ZnSO4⋅7H2O 0.2 mg⋅L–1; CuCl2⋅2H2O 20.0 mg⋅L–1; NiCl2⋅6H2O 20.0 mg⋅L–1; Na2MoO4⋅2H2O 20.0 mg⋅L–1; Na2SeO4, 20.0 mg⋅L–1; Na2WO4, 20.0 mg⋅L–1, and was added at 1 mL⋅L–1medium according to –1. Biomass from a lab-scale chain elongation bioreactor converting lactate and acetate to n-caproate was used as inoculum . Before application, 1 g of nZVI powder was suspended in 4 mL of N2-bubbled demi water. The suspension was mixed for 10 min using a high shear mixer . After this, the bottle containing the slurry was loosely closed to avoid overpressure due to hydrogen gas release and left to rest at room temperature for ∼48 h before adding it to the experiments. This procedure followed the instructions of the supplier and served to erode the protective oxides layer present on the nZVI particles.Lactate used as electron donor in the experiments were: concentrated lactic acid (≥90%) , sodium-D-lactate ≥99.0%  and sodium-L-lactate ≥99.0% . The concentrated lactic acid was a mixture of lactate monomers and oligomers  containi–1 to a 27 g⋅L–1 total lactate (monomers + oligomers) solution prepared with concentrated lactic acid. Hydrolysis was followed by measuring the increase in monomeric lactate concentrations. A control experiment with no nZVI was included. Experiments were done in triplicate at an initial pH of 5.5. Vitamins, yeast extract, (NH4)H2PO4 and trace elements were left out to prevent microbial growth.Incubations were carried out in 125 mL serum bottles using a liquid volume of 40 mL. An overview of the initial conditions in the different experiments can be found in –1) on lactate-based chain elongation was tested in duplicate batch incubations using 30 g⋅L–1 total lactate and 6 g⋅L–1 acetic acid. This resulted in monomeric L-lactate concentrations of 19.3 ± 0.6 g⋅L–1  as expected from 90% concentrated lactic acid solutions  of 0.45 and 1.2 atm. Acetate (5.7 g⋅L–1) and n-butyrate (9.3 g⋅L–1) elongation with H2 as sole electron donor (0.75 atm) was also tested in the same experiment. Lastly, to test the effect of the lactate enantiomer fed and the presence of nZVI on lactate conversion and product spectrum, fermentation of D-lactate and L-lactate with and without nZVI (1 g⋅L–1) was studied in triplicates in experiment IV. Enantiopure or a racemic mixture (1:1) of D- and L-lactate sodium salts were added to reach a lactate concentration of 18 g⋅L–1. This concentration is comparable to the monomeric lactate concentrations measured in the experiments supplied with concentrated lactic acid. Acetate was added as electron acceptor at 5 g⋅L–1. All the experiments were incubated in a shaker at 120 rpm and 30°C. Initial pH was adjusted to 5.5 by adding 4 M KOH and/or 1 M HCl. Headspace gas was exchanged by vacuum/filling cycles (5 times) with N2 in experiment I and N2:CO2 (80:20) in experiments II-IV to a final overpressure of 0.5 atm. N2 was partly replaced with H2 to reach designated PH2 for experiment III. When pressure raised above 2.5 atm during experiments II and III, gas was manually released to bring headspace pressure down to 1.2–1.5 atm and the released gas volume was measured. Gas bags were used in experiment IV to avoid overpressure. Process performance in the incubations was evaluated based on substrate conversion ; lactate enantiomeric distribution; and electron and carbon selectivities  were quantified by gas chromatography. For methods description see 1.Liquid samples were taken regularly to measure pH and soluble metabolites. Samples were centrifuged  and stored at −20°C before metabolites analyses. Lactate (both D- and L-lactate monomers together), succinate and formate were measured by HPLC . Lactate2 under accession number PRJEB41368. DNA sequences were processed as described previously  and used as template for amplifying the V3–V4 region of 16S rRNA via Illumina sequencing using the primer sets described by eviously : the DADeviously  was usedeviously  and visueviously . Correlaeviously .–1 when 1 g⋅L–1 of nZVI was added and from 16.5 ± 1.0 to 24.5 ± 0.3 g⋅L–1 with 5 g nZVI⋅L–1, showing that nZVI is capable to support hydrolysis of lactate oligomers present in concentrated lactic acid solutions  , slightly higher than at 0.5 and 2 g nZVI⋅L–1 . Conversion rates were much lower at higher nZVI doses of 3.5 and 5 g⋅L–1 .Adding nZVI at concentrations ranging from 0.5 to 5 g⋅Lays 0–7) . The hig–1 in the control experiment and were consistently lower in the presence of nZVI. With nZVI concentrations between 0.5 and 2 g⋅L–1, n-caproate production was promoted, reaching n-caproate concentrations of 4.3 ± 0.3 g⋅L–1 at 1 g nZVI⋅L–1 (H2) was observed in all tested conditions for experiment II giving no obvious relation between PH2 and n-caproate formation (–1) or 1.2 atm in experiment III did not result in n-caproate formation. Additionally, neither acetate nor n-butyrate were elongated with hydrogen as sole electron donor . Carbon was diverged toward propionate at low selectivities (2–4% at 0–2 g nZVI⋅L–1) although it raised up to 10 ± 1% and 21 ± 6% when adding 3.5 and 5 g nZVI⋅L–1, respectively. Carbon selectivity for CO2 ranged between 37 and 52% by the end of the chain elongation phase (days 0–7) and showed a decreasing trend during the hydrogen consumption phase (2 carbon selectivity (8–15% lower) was observed in incubations displaying n-valerate and n-caproate formation.The microbiome produced n-butyrate efficiently with an electron selectivity of 91 ± 1% in the control experiment. At 1 g nZVI⋅Lon phase . Evident–1) and without nZVI. Both enantiomers were converted into n-butyrate  with either D-lactate, L-lactate or a racemic mixture were carried out with . Conversion rates on days 3–5 were 4.6 ± 1.2 g⋅L–1⋅d–1 for D-lactate and 7.1 ± 0.7 g⋅L–1⋅d–1 for racemic lactate. n-butyrate production rates for days 0–3 were 0.67 ± 0.35 and 1.62 ± 0.45 for the racemic mixture and D-lactate, respectively. From days 3 to 5, n-butyrate productivities from L-lactate, D-lactate and the racemic mixture were, respectively, 5.34 ± 0.15, 3.0 ± 0.56 and 4.46 ± 0.49 g⋅L–1⋅d–1. The enantiomeric excess for L-lactate was still high (88 ± 7%) on day 3 and D-lactate was measured to be 1.07 ± 0.64 g⋅L–1 . In general, enantiopure lactate (either D- or L-lactate) was racemized during fermentation while racemic equilibrium was maintained when feeding racemic lactate  and Eubacterium species (Clostridium sensu stricto 12 species were enriched (32–47% relative abundance) in n-caproate producing conditions compared to incubations without nZVI (12–16%). This genus was predominantly represented (65–86% of counts) by Clostridium tyrobutyricum sequences (ASV1) which showed relative abundances of 21–32% (samples 0.5–1 g nZVI⋅L–1). Clostridium luticellarii (ASV29) from the same genus was also enriched to relative abundances 3–7% when n-caproate was produced while it remained <1% in the other conditions. Three sequences were classified as Caproiciproducens species of which Caproiciproducens sp. ASV37 was identified only in n-caproate producing incubations (3–4% relative abundance) and showed low similarity to C. galactitolivorans BS-1 (90% identity). Caproiciproducens sp. ASV37 (p < 0.01), Clostridium tyrobutyricum sp. ASV1 (p < 0.001) and Clostridium luticellarii sp. ASV29 (p < 0.001) were positively correlated with n-caproate concentrations. The genus Ruminococcaceae was correlated with both n-valerate (p < 0.01) and n-caproate (p < 0.001). At ≥ 3.5 g nZVI⋅L–1, relative abundance for Clostridium sensu stricto 12 was reduced (8–18% relative abundance) while Clostridium novyi sp. ASV7, only member of the genus Clostridium sensu stricto 7, was enriched to relative abundances of 14–24%  and Sedimentibacter sp. ASV10  reached 17 and 7% relative abundance, respectively. Clostridium sensu stricto 7, Oscillibacter, Oscillospiraceae and Sedimentibacter species were among the highly abundant genera related with high nZVI and propionate production.Composition of microbiomes from experiment II was affected by nZVI presence and concentration . nZVI pr species . Severalf 14–24% . At 5 g Clostridium sensu stricto 12, Haloimpatiens, unclassified Lachnospiraceae and Oscillibacter were negatively related with nZVI (Clostridium sensu stricto 12 species (being > 77% C. tyrobutyricum sp. ASV1) showed decreased relative abundances from up to 75 to <1% with nZVI addition. On the other hand, Sedimentibacter species along with the less abundant genus Caproiciproducens, unclassified Microbacteraceae and unclassified Oscillospiraceae showed a positive correlation with nZVI. Other highly abundant genera such as Proteiniborus and Corynebacterium, with Corynebacterium provencense sp. ASV4 as the only member, showed no significant correlation with nZVI (Eubacterium (p < 0.001), Lachnospiraceae UCG-010 (p < 0.001), Lutispora (p < 0.01) and Methanoculleus (p < 0.0005). Corynebacterium provencense was negatively correlated with acetate formation (p < 0.0005). Methanoculleus sequences  were highly abundant in the inoculum sample and were also detected in the incubations. However, methane was not detected in our experiments and M. palmolei is reported to not grow on lactate  react with lactyl lactate (2 mol lactyl lactate–⋅ Fe0–1) to produce lactate (Eq. 12), 1 g nZVI⋅L–1 could hydrolyze 50% of the lactate oligomers. Instead, hydrolysis was observed at about 30%. nZVI was added in excess at 5 g nZVI⋅L–1 with incomplete hydrolysis obtained despite nZVI corrosion was still feasible at the near-neutral pH observed . Hydrolysis of lactate oligomers was enhanced with nZVI under fermentation conditions probably by a combination of chemical and biological hydrolysis  while n-caproate showed a positive correlation (p < 0.01) (–1 compared to ≥ 3.5 g⋅L–1 (days 0–2). Overall, lactate was depleted within 7–9 days. Under anaerobic conditions, nZVI corrosion occurs rapidly in the first ∼24 h to gradually level out reaching almost full depletion in 2–7 days depending on the system conditions  compared to lactate (Eqs. 4–8) or hydrogen (Eqs. 17 and 18), lactate was available at higher amounts with nZVI being a secondary electron source. Considering that bacteria may derive electrons from the conversion of lactate to acetate  to drive the RBO pathway, Fe0 in added nZVI  could have contributed to 1–10% (0.5–5 g nZVI⋅L–1) of electrons in total lactate. Both n-valerate and n-caproate production correlated (p < 0.01) with increased substrate conversion  . Moreovenditions . The assnditions  and incrnversion  indicatinversion . Severalutyricum , C. luticellarii  and memboccaceae  includines e.g.,  were enrproducer  but is ncellarii , Ruminococcaceae , and Caproducens  organism–1), other potential n-butyrate producers such as Oscillibacter, unclassified Oscillospiraceae and Sedimentibacter species were enriched. These taxa have also been enriched in bioelectrochemical systems  (H2) did not seem to influence lactate chain elongation or propionate formation since similar PH2 values and behavior were observed with and without nZVI addition in experiment II. Other reports show improved lactate conversion to MCC with hydrogen addition (H2 up to 2.6 atm (experiment II) were observed. H2 addition in experiment III did not alter product spectrum from lactate and H2 did not elongate acetate or n-butyrate in the absence of lactate. Although not tested in this study, acetate and n-butyrate elongation with nZVI (Eqs. 15 and 16) may be more feasible than with H2 as the electron donor (Eqs. 17 and 18). At high nZVI concentrations (5 g⋅L systems  and Osciith nZVI . Howeverith nZVI . Clostriormation . C. novy2 uptake  which mi pathway . Propion< 0.001) . Here, naddition . This waM. elsdenii is reported to produce even-chains from D-lactate and propionate/odd-chains from L-lactate  complex] to drive the endergonic reduction of NAD  by simultaneously oxidizing Fdred  of electrons in total lactate. However, when considering the confurcating lactate oxidation to pyruvate step , nZVI contributions through Fdred would be doubled. Therefore, small amounts of nZVI could significantly influence lactate-to-pyruvate flux and the formation of elongated products. nZVI could also influence energy conservation and butyryl-CoA dehydrogenase in the RBO pathway .nZVI has been shown to decrease ORP creating favorable conditions for ethanol-based chain elongation . The samng Fdred . Under aeduction . H2-meditabolism . Reverserm Fdred  being ththan Ech . As ment pathway . ExtraceClostridium (C. tyrobutyricum) and unclassified Lachnospiraceae being outcompeted by other potential n-butyrate producing bacteria. Sedimentibacter isolates are reported to use pyruvate and amino acids, but not lactate, with production of SCC, mainly acetate and n-butyrate without H2 formation . Genes involved in the RBO pathway are also present in C. provencense and other Corynebacterium species (Proteiniborus isolate from anaerobic digestion was predicted to harbor lactate dehydrogenases and produce acetate (including Wood-Ljungdahl pathway), propionate (acrylate pathway) and n-butyrate (butyrate kinase pathway) (+ levels between experiment IV using sodium lactate (∼4.8 g Na+⋅L–1) and experiment II using concentrated lactic acid (∼0.13 g Na+⋅L–1)  may have contributed to relatively different microbiomes developed in the two experiments.The addition of nZVI to fermentation of both enantiopure and racemic lactate substrates resulted in faster lactate conversion to n-butyrate. Notably, the microbiome composition was drastically changed by nZVI addition with ormation . Some st systems  and grap systems . Additioeduction . Therefo species . A Protepathway) . DiffereAlthough nZVI may inactivate gram-negative more severely than gram-positive bacteria , taxa en2 (Eq. 14). Hydrogen consumption was observed in the last days of incubation in experiments II (days 7–12) and IV . However, different substrate conversions observed in experiment II affected apparent H2 and CO2 formation/consumption during this phase. No significant correlation was found between H2 consumption and CO2 or acetate formation during the hydrogen consumption phase but acetate formed during this phase was positively related with substrate conversion, n-valerate and n-caproate concentrations  . This indicates that CO2 may have been recovered with nZVI or H2. Decreases in CO2 carbon selectivity during the hydrogen consumption phase were related with higher substrate conversions (p < 0.001) (toward chain elongation) and less CO2 release (p < 0.01)  was observed for C. luticellarii which may use the Wood-Ljungdahl pathway to take up electrons, H2 and/or CO2 coupled to chain elongation  and CO2 (p < 0.05) consumption during the hydrogen consumption phase  or H < 0.01) . This su < 0.01) . CO2 couongation . Caproicon phase . Of the oduction . Overallacterium  were ide–1 (<0.5% of iron added) in all the incubations with nZVI. Thus, we could not quantify the extent of nZVI reaction based on soluble iron. The formation of insoluble iron species may account for such low soluble iron concentrations.Soluble iron concentrations by the end of the experiments were below 3 mg⋅LClostridium acetobutylicum converting lactate and acetate to n-butyrate  while K-strategists exhibiting high-yield metabolism  thrive under substrate-limiting conditions (tyrobutyricum outcompeting Caproiciproducens-related species that were highly abundant in the inoculum derived from a continuous chain elongation reactor (section “nZVI Shapes Microbiome Composition”). Presence of n-butyrate favors n-caproate producers in (repeated) batch reactors fermenting food waste .Even though lactate-to-acetate ratios tested here could in principle yield n-caproate , Eq. 8, butyrate . This exbutyrate . n-butyrbutyrate , it requbutyrate . Based onditions . Followiod waste  which isod waste . Despiteod waste , growth od waste  and highC. novyi. In rumen fermentation, where the acrylate pathway is dominant, an excess of D-lactate was also associated with higher propionate formation , a racemic mixture of lactate might result in higher chain-elongating rates in the case that both enantiomers are metabolized simultaneously. However, we observed higher conversion rates with enantiopure substrates [D-lactate (days 0–3) and L-lactate (days 3–5), section “Experiment IV—Lactate Enantiomers Conversion to n-Butyrate at Different Rates Boosted by nZVI”). Whether D- or L-lactate was the required substrate for chain elongation is not clear from our experiments due to the observed lactate racemization. The n-caproate producer Rumminococcaceae bacterium CPB6 possess both L-Ldh and D-Ldh and the expression of L-ldh in recombinant E. coli was shown to enhance lactate conversion by 17-fold , Corynebacterium, Eubacterium, Haloimpatiens, Lachnoclostridium, Lachnospiraceae, Oscillibacter, Rumminococcaceae (including Rumminococcaceae bacterium CPB6) and Sedimentibacter. Other genera include the PAB Propionibacterium and Veillonella  through reductive calcination  . Therefore, cathodic electrons could be continuously supplied under controlled pH conditions to steer propionate formation and chain elongation of even or odd carboxylates.Reaction rates and MCC selectivity observed here could be improved by testing nZVI in pH-controlled experiments. Mildly acidic conditions not only favor both iron corrosion and chain elongation but may decrease the likelihood of iron precipitates formation  and allocination . Soluble (FeOOH)  and may  (FeOOH) . Importanditions  where reLactate-based chain elongation microbiomes, lactate conversion rates and product spectrum were affected at different nZVI doses. The effects of nZVI on chain elongation were related to hydrolysis of lactate oligomers, changes in pH and possibly by acting as additional electron donor. The metabolism arising under specific conditions  determined both lactate enantiomeric proportions and product spectrum. Lactate was racemized in chain-elongating microbiomes while D-lactate excess was observed during propionate production through the acrylate pathway. Our results suggest that feeding D-lactate to continuous reactors would not necessarily translate into higher chain elongation rates due to lactate racemization. Carbon recovery into carboxylates was increased after hydrogen consumption which was presumably coupled with carbon dioxide recapture into acetate and elongated carboxylates. Fermentation conditions imposed by the presence and concentration of nZVI could be replicated with bioelectrochemical systems to control carboxylates production under continuous operation.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CC-D designed the study, carried out the data analysis, and drafted the manuscript. JE carried out the experiments and supported the data analysis. CB supported in the review and editing of the manuscript. DS conceptualized, designed and supervised the study, and supported in the review and editing of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hif3α in murine pre-adipocyte 3T3-L1 cell line, a versatile and well characterized biological model of adipogenesis, by gain- and loss-of function approaches and in thermogenesis-induced model in vivo. HIF3A is regulated by inflammation, it modulates lypolysis in adipose tissue of obese adults, but its role in energy metabolism has not previously been investigated. We characterized gene and protein expression patterns of adipogenesis and metabolic activity in vitro and mechanistically in vivo. Overexpression of Hif3α in differentiating adipocytes increases white fat cells, whereas silencing of Hif3α promotes “browning” of white cells, activating thermogenesis through upregulation of Ucp1, Elovl3, Prdm16, Dio2 and Ppargc1a genes. Investigating cell metabolism, Seahorse Real-Time Cell Metabolism Analysis showed that silencing of Hif3α resulted in a significant increase of mitochondrial uncoupling with a concomitant increase in acetyl-CoA metabolism and Sirt1 and Sirt3 expression. The causal Hif3α/Ucp1 inverse relation has been validated in Cannabinoid receptor 1 (CB1) knockout, a thermogenesis-induced model in vivo. Our data indicate that Hif3α inhibition triggers “browning” of white adipocytes activating the beneficial thermogenesis rewiring energy metabolism in vitro and in vivo. HIF3A is a novel player that controls the energy metabolism with potential applications in developing therapy to fight metabolic disorders, as obesity, T2D and ultimately cancer.Maintenance of energy balance between intake and expenditure is a prerequisite of human health, disrupted in severe metabolic diseases, such as obesity and type 2 diabetes (T2D), mainly due to accumulation of white adipose tissue (WAT). WAT undergoes a morphological and energetic remodelling toward brown adipose tissue (BAT) and the BAT activation has anti-obesity potential. The mechanisms or the regulatory factors able to activate BAT thermogenesis have been only partially deciphered. Identifying novel regulators of BAT induction is a question of great importance for fighting obesity and T2D. Here, we evaluated the role of  The frequency of metabolic disorders, such as obesity and type 2 diabetes (T2D) rose up over the last 50 years and the generation of novel therapeutic approaches aimed to ameliorate the balance of energy metabolism in adipose tissue is going to be developed . The adiHIF1A and HIF2A) are involved in metabolic changes driving cell adaptation to low oxygen availability , suggesting that it was involved in sensing inflammation rather than hypoxic stress  mouse, an established thermogenesis-induced model , dexamethasone (1uM) and 3-isobutyl-1-methylxanthine  on post-confluent cells for 3 days, then changed to DMEM plus insulin (10 ug/ml). Different cytokines were added on post-confluent cells, according to experimental design .Grown and differentiation of 3T3-L1 cells were previously described . BrieflyLipid accumulation was visualized by light microscopy in immature and mature adipocytes plated in 12-well plates at differentiation days 3, 7 and 10. Cells were fixed with 4% formalin in PBS for 1 h and rinsed with distilled water at RT. The fixed cells were treated with 60% isopropyl alcohol for 5 min and then stained with 0.3% Oil Red O in 60% isopropyl alcohol solution for 15 min at RT and subsequently washed with distilled water. Accumulation of lipids was quantified by isopropanol extraction for 15 min and absorbance measured with Infinite M1000 microplate reader (TECAN) at 540 nm.5 cells/well density. After 24 h, cells were transfected with 50 nM of small interfering RNAs (siRNAs) targeting Hif3α  or non-targeting siRNA pool for silencing experiments. Overexpression experiments were conducted using the pM02 plasmid containing the ORF of HIF3A (Z1165) purchased by GeneCopoeia with a CMV promoter. Both experiments were performed using Lipofectamine 2000 , according to the manufacturer’s protocol. The day after transfection, cytokines were added in the differentiation medium and cells harvested at differentiation days 3 and 7.3T3-L1 cells were seeded into 6-well plates at 3 × 10(−ΔΔCt) method and exhibited as heatmap by R package.Total RNA was isolated from mouse FAT and from 3T3-L1 cells using TRIzol Reagent  following the manufacturer’s instructions. In brief, the mouse samples were homogenized in TRIzol Reagent (1 ml/100 mg of tissue); then samples (mouse and cells) were incubated for 5 min at 20°C for the dissociation of nucleoprotein complexes and added 0.2 ml chloroform/ml TRIzol Reagent following centrifugation at 12000xg for 15 min at 4°C. The aqueous phase containing the total RNA was precipitated by mixing isopropyl alcohol (0.5 ml/ml TRIzol Reagent) at −20°C O.N. After centrifugation at 120,00 x g for 10 min at 4°C, the pellet of RNA was washed with 75% ethanol, centrifuged at 7,500 x g for 10 min at 4°C and dissolved in an appropriate volume of DEPC water. The quantity and the purity of total RNAs were measured by NanoDrop 200 spectrophotometer . Then 1 ug/ml were converted to cDNA by reverse-transcription using Luna Script RT Super Mix Kit (Biolabs E3010). qRT-PCR was performed using SybrGreen PCR Master mix 2x reagent in the CFX96TM Real Time PCR Detection Systems . Gene expression was normalized to the internal references’ genes (18S and β-actin). Relative expression was calculated using 22, 1% NP-40, 10% glycerol, 0.1 M NaF, 100 μM Na3VO4 and protease inhibitors mixture . Equal amounts of proteins were separated by SDS-polyacrylamide gels and transferred to nitrocellulose membranes . Membranes were incubated with blocking buffer (TBS-Tween buffer containing 5% milk) for 1 h at RT and subsequently with primary antibodies (see below) at 4°C ON. After three washes for 10 min with T-TBS buffer , the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody  for 1 h at RT and then washed for 15 min with T-TBS buffer. The resulting immunocomplexes were detected using Amersham ECL Plus .Cells were lysated in 20 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl4 cells/well of 3T3-L1 cells were plated on a Seahorse XF24 cell culture microplate and transfected with siHif3α. At 24 h, the medium was replaced with differentiated medium in presence of pro-inflammatory cytokines. After 72 h, the plate was loaded into the by Seahorse XF24 Analyser (Agilent Technologies) and analysed as reported in , according to the manufacturer’s protocol. Briefly, 2×10orted in . MultiplGuide for Care and Use of Laboratory Animals . Mice were housed in a specific pathogen-free facility, kept under controlled environmental conditions , maintained on a standard diet with free access to water. Heterozygous males and females (CD1 strand) carrying a Cb1 null deletion  with 4 µl of Proteinase K  and digested at 600 rpm at 56°C ON. The samples were then heated at 95°C for 15 min to inactivate proteinase K and were briefly centrifuged at 13,000×g for 1 min. After centrifugation, genomic DNA (gDNA) was isolated by the traditional phenol/chloroform method and the amount of gDNA determined by spectrophotometry . The purity of gDNA was determined by ratio A260/A280 = 1.8.The genotype was determined by PCR analysis of gDNA using specific primers for neomycin-cassette  and CB1 gene as already reported [12], at the following conditions: 94°C for 5 min, 1 cycle; 94°C for 1 min, 56°C for 1 min, 72°C for 1 min, 35 cycles; and 72°C for 5 min, 1 cycle. Finally, 25 μl of PCR amplification mixtures were analysed by electrophoresis on 1.3% agarose gel and stained with 0.5 μg/ml of ethidium bromide.vFAT and iFAT mass from WT and CB1KO animals were fixed in Bouin’s solution for 12 h, dehydrated in ethanol, cleared in xylene and embedded in paraffin. Sections (13 μm) were cut and processed for haematoxylin-eosin stain and used for adipocyte count, as already reported .i.e., number of adipocytes per area) was analysed. Random not serial sections (n = 10/genotype) were used to capture the images  by a high-resolution digital camera . The density of adipocytes was calculated per optical field. In particular, for each section a minimum of 20 random optical fields were selected and subjected to count of adipocytes. Results were expressed as mean value of number of cells per 100 mm2 ± SEM.Stained sections were early observed under a light microscope  then density of fat mass , anti Gapdh  anti Tubulin (Santa Cruz Biotech #5286), and rabbit polyclonal anti LC3-I/II (abcam #41420), anti AceCs1 , anti Acc , anti PDH , anti Sirt3 (abcam #40963–100), anti Hif3α (Proteintech #27650-1-AP) anti Actin (Santa Cruz Biotech #1615) were used for western blotting. The oligonucleotides used in this study are reported in p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001.All tests were executed in triplicate and repeated three times. Standard error of mean (SEM) bars was reported. Student’s t-test was performed: ∗HIF3A expression in an oxygen-independent manner in MSCs [8], therefore, we asked whether the cytokine-mediated effects could regulate fat metabolism and differentiation. The preadipocyte 3T3-L1 cells were exposed to pro-inflammatory cytokines  and angiogenic markers (EGF and VEGF), which are secreted by hypercaloric state adipocytes and angiogenesis, respectively  and Hif3α knocking down (siHif3α) cells, were generated and the impact of Hif3α deregulation characterized  were induced during adipocyte differentiation, in WT and TO cells, and almost all pro-inflammatory cytokines enhanced its expression  cells, bolished , and itsar model .Pparg), the CCAAT enhancer binding protein alpha (c/Ebpα) and the C-type lectin domain containing 10A (Clec10a) and genes involved in lipid storage or energy supply as Cd36 molecule (Cd36) and fatty acid synthase (Fasn). The experiments were performed in overexpressing (HIF3A-TO) and downregulating (siHif3α) cells and compared to wild type cells (WT) in immature (T0) and mature adipocytes (T3 and T7)  . The hea markers . In addia HIF3A. . To notea HIF3A. , this rePparg, c/Ebpα, Clec10a, Cd36 and Fasn) in siHif3α cells impacted the adipocyte phenotype. Cells were induced to differentiate and mature adipocytes stained with Oil Red O and quantified. A marked decrease in lipid abundance is observed in siHif3α mature adipocytes, compared to the control (Ucp1), elongation of very long chain fatty acids 3 protein (Elovl3), positive regulatory domain containing 16 (Prdm16), type II iodothyronine deiodinase (Dio2) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a). Heatmap showed that the expression of these markers was massively increased in siHif3α-derived adipocytes, suggesting a transition toward “brown/beige” phenotype (Hif3α mediates the appearance of BAT-like adipocytes via Ucp1 upregulation and a consistent downregulation of WAT markers.Lipid droplets (LD) are not only a passive storage of lipids, but dynamic organelles that play a role in energy metabolism. We evaluated if and how the consistent downregulation of WAT markers . In conditional Angplt4KO cells, the browning of WAT adipocytes is evident, increasing fatty acid oxidation (Hif3α on Angplt4 expression. Angplt4 was induced during WAT differentiation in WT adipocytes (HIF3A further increased Angplt4 mRNA (grey bars). Cytokines pre-treatment had any significant effect on Angplt4 at T0 compared to untreated cells and only a mild additional effect during differentiation (T3 and T7), whereas Angplt4 was nearly absent in siHif3α adipocytes (light grey bars) and the cytokines were unable to rescue its expression, suggesting that the lypogenesis is reduced and, more important, we confirmed a transcriptional control of Angplt4 by Hif3α (Reduced lipid catabolism/degradation (lypolysis) can lead to accumulation of lipids (lipogenesis), adipocyte hypertrophy and storage in lipid droplets (LD), which are related to insulin resistance and increased inflammation. Lipoprotein lipase (LPL) catalyses the hydrolysis of triacylglycerol into glycerol and fatty acids (TAG), whose activity is inhibited at a posttranscriptional level by activation of the angiopoietin-like 4  and proton leak compared to WT, showing increased mitochondrial uncoupling, consistent with the characteristics of brown/beige adipocytes . De facto, the baseline OCR/ECAR ratio indicated that the stressed ECAR parameter including both glycolysis and mitochondrial activity for each cell type was due to the prominent contribution of CO2 to ECAR, mainly in siHif3α adipocytes  (pyruvate to acetyl-CoA), acetyl-CoA synthetase (AceCs1) (acetate to acetyl-CoA) and acetyl-CoA Carboxylase (ACC)  that provide acetyl-CoA for catabolic (TCA cycle), and/or anabolic (fatty acid and cholesterol synthesis) purposes. Western blotting showed that the adipocytes in absence of Hif3α (siHif3α adipocytes) showed an increase of AceCS1 and PDH enzymes and downregulation of ACC .The large amount of energy activated in absence of n of ACC . Thus, ivia the epigenetic regulation of gene expression  indicated that adipocyte density was significantly higher (***p < 0.001) in CB1KO, both in vFAT and iFAT compared with WT, demonstrating an effect of CB1KO on fat cell density   . Hif3α med to WT , confirmHif3α plays a role in adipogenesis controlling WAT differentiation. The inhibition of Hif3α unexpectedly promoted the “browning” of white adipocytes, with an increase in energy metabolism via Ucp1-mediated thermogenesis in vitro and in thermogenesis-induced model in vivo. Thus, Hif3α is a regulator of BAT induction, guiding beneficial metabolic reprogramming, dissipating energy as heat thereby improving glucose and lipid metabolism, decreasing the insurgence of metabolic disorders.Taken together, our results showed that The causal mechanisms underlying adiposity and metabolic disorders remain unclear.Hif3α might control the adipocyte physiology in a dynamic manner: Hif3α senses and drives metabolic changes to adapt to inflammatory status that promotes lipid accumulation; if the stressor is removed, the cellular homeostasis can be rescued, but in chronic conditions it becomes maladaptive and the adipocytes start to accumulate lipids, expanding WAT.We speculated that HIF3A is methylated and this modification is significantly associated to obesity  increases risk of obesity and diabetes. HIF3A remained methylated in hyperglycemic mothers, whereas is active in normoglycemic ones. Methylation of HIF3A locus is also modifiable by diet. Huang et al. found that vitamin B and folate intake influences the methylation of a specific SNP in HIF3A locus  and/or cell number (hyperplasia) . In huma3A locus , indicat3A locus . In linechanisms .In other cells of the body, the balance between metabolites and chromatin remodelling coexist as in immune cells, in which malonyl-CoA for histone malonylation , lactateHIF3A expression in mature and precursor adipocytes and knock-down of HIF3A in human MSCs increased fatty acid oxidation, suggesting that reported studies and our results support the hypothesis that HIF3A is relevant in adipogenesis and methylation is secondary to disrupt the metabolism leading to obesity.Therefore, obesity can be considered an epigenetic disorder, in which DNA modifications  are the mechanism by which the genome of adipocytes can be modified, predisposing to later disease risk . Genome-HIF3A by Gene Expression Profiling Interactive Analysis (GEPIA) in cancers mostly associated to obesity : HIF3A is significantly downregulated in tumor vs. normal tissue and the promoter regions are methylated (To note, epigenetic deregulation is also a hallmark of cancer initiation and progression and it is now clear that obesity increases the risk to develop a cancer. As speculation, we looked at the expression data of thylated , suggestHIF3A is a strong candidate gene with novel regulatory function in fat metabolism and can pave the way for developing metabolic therapy to target the abnormal energy intake in adipocyte cells, enhancing human health.Future studies are needed to understand the dynamics of this metabolic and epigenetic crosstalk, but this report provides evidence that


The magnetization saturation and BET surface area for Fe3O4, Fe3O4/C, and α-Fe2O3/C were measured as 90, 86, and 27 emu/g and 16, 56, and 89 m2/g with an average pore size less than 7 nm. Cyclic voltammogram and galvanostatic charge/discharge studies showed the highest specific capacitance of carbon-modified Fe3O4 and α-Fe2O3 as 213 F/g and 192 F/g. The in vivo biological effect of altered physicochemical properties of Fe3O4 and α-Fe2O3 was assessed at the cellular and molecular level with embryonic zebrafish. Mechanistic in vivo toxicity analysis showed a reduction in oxidative stress in carbon-modified α-Fe2O3 exposed zebrafish embryos compared to Fe3O4 due to despaired influential atomic interaction with sod1 protein along with significant less morphological abnormalities and apoptosis. The study provided insight into improving the characteristic of MNPs for electrochemical application and higher biological biocompatibility.Recent advancement in nanotechnology seeks exploration of new techniques for improvement in the molecular, chemical, and biological properties of nanoparticles. In this study, carbon modification of octahedral-shaped magnetic nanoparticles (MNPs) was done using two-step chemical processes with sucrose as a carbon source for improvement in their electrochemical application and higher molecular biocompatibility. X-ray diffraction analysis and electron microscopy confirmed the alteration in single-phase octahedral morphology and carbon attachment in Fe  •Carbon modification of octahedral shaped magnetic nanoparticles (MNPs) was done using sucrose.•3O4, Fe3O4/C and α-Fe2O3/C was 90, 86, 27 emu/g and 16, 56, 89 m2/g.The magnetization saturation and BET for Fe•2O3 induce less ROS in exposed zebrafish embryos.Carbon modified α-Fe•3O4 despair influential atomic interaction with zebrafish Sod1 protein.Carbon modified Fe•3O4 exhibit less morphological abnormalities and apoptosis.Carbon modified Fe The u3O4, V oxide-based materials, and NiO-based materials for the corresponding applications. However, these materials have limitations such as low theoretical capacitance value 1,100–1,300 F/g in case of Ru-based materials [3O4 and α-Fe2O4 have been found as suitable for PC electrode materials due to the higher theoretical capacitance value as 2,606 and 3,625 F/g [3O4 was solved by combining it with electric double layer capacitor (EDLC) materials. As a result, the capacitive characteristics were found enhanced by combining Fe3O4 and α-Fe2O3.An enormous amount of research effort has been put to improve the electrochemical properties of supercapacitor materials. The major effort has been put in improving the capacitance and the cycle stability to select the appropriate electrode materials in supercapacitors with an electrical double layer, pseudo-capacitive, and their combination materials ,14. The aterials , low cheaterials ,18, and aterials  and NiO-aterials . Fe oxid,625 F/g . The proA part from iron oxide-based materials, carbon-based materials have also been recognized as suitable materials to prepare the hybrid electrode material  due to its easy preparation process, lower cost, larger surface area, and good electronic conductivity ,21. Carb3O4-based nanoparticles have been reported for their toxicological effects on different cell lines [3O4-based nanoparticles will have a synergistic and reduced toxicological impact on biological entities due to alteration in electrochemical properties of Fe3O4 by carbon modification. The hypothesis has been investigated in this report by evaluation of biocompatibility of synthesized nanomaterial with embryonic zebrafish model [Concerning the modern scenario of environmental sustainability, the eco-compatibility of any new nanomaterials has been a crucial factor for its applicability. The utility of the material during the usage as well as after-usage has to be considered in a limited way, provided their extent of effects on environment and biological identities. The deployed nanomaterials especially MNPs like iron oxide can be accumulated in the environmental sources after being used. These accumulated nanomaterials can further leak on abiotic resources like water, soil and show their impact on aquatic animals affecting the aquatic ecosystem. Hence, it is important to investigate the eco-compatibility of new synthesized material. Fell lines  as well ll lines  and zebrll lines . Carbon-ll lines . Hence, sh model . Moreove22.13O4 MNPs were prepared by alkaline precipitation  [2.4H2O (3.976 g) was dissolved in double distilled water and stirred at 400 rpm using magnetic stirrer. At 80 °C, NaOH (0.2 M (1.6 g)) solution was added drop wise for the reaction process and then KNO3 solution was added for the oxidation process. NaOH was used for the metal hydroxide precipitation and KNO3 was used to oxidize the ferrous ions to the ferric state. The reaction was done in 200 mL of double distilled water. The solution was stirred at 90 °C for 2 h. The precipitates were collected using magnetic separation process and washed several times to remove residual unreacted products. The collected black powders were dried at 90 °C in the hot air over for 12 h.Fe method) . In brie3O4 MNPs were dispersed in the sucrose solution and sonicated to achieve the uniform dispersion of nanoparticles. The dispersion was further heated to 90 °C to convert the solution into semisolid. Furthermore, the mixture was heated to 200 °C for 12 h for the pyrolysis. The mixture was further heated at 600 °C and 800 °C for 2 h in nitrogen atmosphere. Followed by heating and cooling, the carbon-modified MNPs were washed several times with double distilled water and collected using magnetic separation process to remove excess carbon nanostructures unattached with MNPs. Final powders were dried at 80 °C for 4 h and named as F1, F2, and F3 corresponding to bare Fe3O4, carbon modified with 600 °C and 800 °C.For the carbon modification, 1 g of sucrose was mixed with double distilled water and stirred continuously until to get the clear solution. Then, 1 g prepared Fe2.22 and kept at room temperature for slow evaporation. The cyclic voltammetry (CV) analysis was performed with a potential widow of −0.8 to 0.3 V vs. Ag/AgCl at various scan rate of 2–200 mV/s. A platinum (Pt) wire, Ag/AgCl (sat. KCl) electrode, and prepared composite-coated GCE were used as counter, reference, and working electrodes respectively. The specific capacitance was calculated from the galvanostatic charge/discharge at different current densities ranging from 0.5 to 20 A/g in the potential range of −0.1 to 0 V. The electrochemical impedance spectroscopy (EIS) analysis of the electrodes was studied with a perturbation of sinusoidal voltage signal of 10 mV applied over wide range of frequency between 1 MHz and 0.01 Hz with open circuit potential. The zeta potential was determined by using Zetasizer .The synthesized MNPs were analyzed by X-ray diffraction (XRD) by Bruker D8 X-Ray diffractometer with a Cu K source. The morphology analysis of the MNPs was done with field emission scanning electron microscopy (FESEM) (Carl Zeiss Neon 40) and transmission electron microscopy . Room temperature magnetic properties were analyzed by using vibrating sample magnetometer . All the electrochemical measurements were carried out by using CHI760E bipotentiostat  with KOH (6 M) solution. Ten milligram of prepared particles was mixed in 5 mL of stock solution , 79.6 mL Milli-Q water, and 0.4 mL of Nafion) by sonication for 2 h. Solution (5 μL) was coated on a cleaned glassy carbon electrode (GCE) with an area of 0.0707 cm2.33, 18 g sea salt, and 8.4 g CaSO4 per 1,000 mL. Nutritional regulation was done by providing feed three times in a day with blood worm containing fish food. The culturing and obtaining of embryos were done in breeding setup with net partition and breeding female:male in 2:1 ratio. Photoperiodism was maintained as per the standard regulation of 12 h dark and light respectively. Viable embryos were separated from non-viable ones after obtaining embryos and rinsing with the medium. All the experiments were performed in filter sterilized Holtfreter medium [All animal procedures were agreed by the relevant guidelines of Institutional Animal Ethics Committee (IAEC) of KIIT University. All experiments were done in accordance with relevant animal practice guidelines and regulations of IAEC, KIIT University. The adult zebrafish rearing and maintenance was achieved in an overflow container setup supplied by Aquaneering, USA. Equilibration of the system was done with fish water containing 75 g NaHCOr medium . Chemica2.43O4 and α-Fe2O3 was performed with 3- to 4-h post fertilized (hpf) embryos at blastula stage as mentioned by Verma et al. [3O4 and α-Fe2O3/C for 72 h in 24-well plates with Holtfreter medium (HF) buffer. The experimental setup was incubated in 14/10 h light/dark at 28 ± 1 °C. Unexposed embryos were taken as control. The morphological changes were analyzed using stereomicroscope. Different morphological defects like notochord developmental defects, pericardial edema, and the swollen yolk were observed in the exposed embryos. Hatching rate was determined as ratio of embryos hatched by 72 h after fertilization compared to the untreated group. Mortality rate was evaluated as the percentage ratio of number of viable embryos compared to untreated one 72 h after fertilization. Heartbeat count per minute was taken to assess the heart rate. All the experiments were done in triplicates and repeated thrice.Comparative biocompatibility assessment of carbon-modified Fea et al.  and accoIn vitro biocompatibility assessment of Fe3O4 and α-Fe2O3/C was also performed with colon cell lines (HCT116) by evaluating the survivability of cells on exposure to Fe3O4 and α-Fe2O3/C. The survivability assay was performed by the 3--2,5diphenyltetrazolium bromide (MTT) assay as mentioned in 2.53O4 and α-Fe2O3/C exposed embryos was done by staining of treated and untreated embryos using 1.25 mg/L 2′,7′-dichlorofluorescin diacetate (H2DCFDA) dye for 20 min in dark [Oxidative stress analysis in Fe in dark . The emb2.63O4 and α-Fe2O3/C was checked by real-time (RT) PCR analysis. To check the gene expression in zebrafish cells, cell suspension was obtained from the embryos treated with 25, 50, and 100 μg/mL for 72 h. The RNA was isolated using TRIzol reagent  according to the manufacturer’s instructions. Consequently, RNase-free DNase I (Fermentas) treatment was done, and complementry dioxyribonucleic acid (cDNA) synthesis was performed using the Hi-cDNA Synthesis Kit . The analysis of RNA quantity and purity was done at each point by Nanodrop (Colibri Microvolume Spectrometer). Quantitative polymerase chain reaction (qPCR) was performed for each sample using the KAPA SYBR FAST qPCR Master Mix (2×)  with a suitable cDNA dilution as a template. To normalize the tested genes’ expression (sod1), 16S (Housekeeping gene) was taken as an internal control. The determination of values of mean fold change expression values was done for three replicates. The primers used are listed in The alteration in expression of sod1 protein in embryonic zebrafish due to exposure of Fe2.73O4 and α-Fe2O3/C were stained with 5 μg/mL acridine orange (AO) dissolved in HF for 20 min followed by washing with HF buffer for removal of extra stains. Images were taken using the green channel of EVOS inverted fluorescent microscope (ThermoScientific) for comparing the apoptosis that occurred in zebrafish embryos due to exposure of different concentration. Data analysis and presentation was done with the help of ImageJ. All the experiments were performed in triplicates and statistical analysis was performed using GraphPad prism 6.Apoptosis analysis of exposed and unexposed embryos was done by Acridine orange staining as described by Verma et al. . In brie2.8Danio rerio) superoxide dismutase (sod1) has been retrieved from UniProt database bearing ID O73872 and subjected for homology modeling using SWISS-MODEL server. Active site pockets have been identified using CastP server. The α-Fe2O3 and Fe3O4 nanoparticles have been modeled using Crystal Maker software using the parameters, e.g., α-Fe2O3 unit cell with R 3̅ c space group and lattice parameters of a = b = 5.0380 Å and c = 13.7720 Å and Fe3O4 unit cell with F d 3̅ m space group and lattice parameters of a = b = c = 8.3941 Å. The modeled nanoparticles and the protein target were subjected for molecular docking using AutoDock Vina [The protein sequence of zebrafish  was observed for the samples calcined at 800 °C. Some minor peaks of α-Fe2O3 were observed for the sample calcined at 600 °C. The average grain size of Fe3O4 and α-Fe2O3 was estimated as 34 nm and 26 nm. The reduction in the grain size for α-Fe2O3/C is due to the smaller particle’s formation from the bigger particle during the phase transformation at higher calcination temperature. There were no characteristic reflections of any carbon allotropes in both the samples, which indicated that the carbon fraction might be less, or in amorphous form. Furthermore, to evaluate the morphology, the prepared samples were analyzed by the FESEM analysis. 3O4 and α-Fe2O3. An interconnected octahedral-shaped morphology was observed with a wide range of particle sizes ranging from 26 to 220 nm. The octahedral morphology was formed without using any surfactant which might be due to the usage of chloride as a starting material. Moreover, the presence of OH− ions/high chemical potential can be attributed to provide the thermodynamically favorable conditions for the particle shape [2O3 was less compared to carbon-modified Fe3O4 which might be due to the higher temperature after heating. 3O4 and α-Fe2O3. The octahedral-shaped particle looks like a cubic shape in different sizes. The morphology changes for the sample calcined at 800 °C is observed. The images were in correlation with the FESEM images. The morphology of the interconnected octahedral shape become partially spherical due to the high calcination temperature. The attachment of carbon materials on the MNPs and the fringes of MNPs are clearly seen from the HRTEM micrograph as shown in 3O4 compared to α-Fe2O3, which supports the FESEM micrograph results. Furthermore, the prepared samples were undergone to VSM analysis for their magnetic characteristics. 3O4, Fe3O4/C, and α-Fe2O3/C. All the three samples exhibited ferromagnetic behavior. A higher saturation magnetization of 90 emu/g was observed for the bare Fe3O4 nanoparticles while it was reduced to 87 emu/g in case of carbon-modified Fe3O4 nanoparticles. The observed saturation magnetization value was nearly equal to the saturation magnetization value of bulk Fe3O4 [3O4 can be attributed to the fraction of non-magnetic carbon attachment on the MNPs. The F3 sample of carbon-modified α-Fe2O3 showed drastic reduction in the saturation magnetization to 24 emu/g. This can be reasoned to the phase change of Fe3O4 to α-Fe2O3 due to higher calcination temperature and the non-magnetic fraction of carbon materials along with the MNPs. The coercivity of bare Fe3O4, Fe3O4/C, and α-Fe2O3/C was observed as 137, 139, and 251. The higher coercivity for α-Fe2O3/C compared to other two samples was reasoned to the phase change at higher calcination temperature.The evolution of the materials for sustainable energy application demands study on materials with higher output and eco-compatibility. This study is intended toward the fabrication of an eco-compatible hybrid magnetic nanomaterial of Felk Fe3O4 . The red2 adsorption/desorption isotherm curve of (a) Fe3O4, (b) Fe3O4/C, and (c) α-Fe2O3/C. Inset shows the corresponding pore size distribution. The BET surface area of bare and carbon-modified Fe3O4 was measured as 16 and 56 m2/g. Bare Fe3O4 exhibited Type II isotherm which belongs to the microporous characteristics of the nanoparticles. The curve at P/P0 = 0.0–0.2 indicates the completion of monolayer and the initiation of multilayer adsorption. The carbon-modified Fe3O4 and α-Fe2O3 exhibited Type III isotherm shape with some area within the loop. The loop was similar to the H3-type hysteresis. As the loop did not show any limit or saturation at higher P/P0, H3-type hysteresis indicated to the aggregation of the particles. Carbon-modified α-Fe2O3 showed the highest surface area of 89 m2/g among the three samples. The average pore size of bare Fe3O4, carbon-modified Fe3O4, and carbon-modified α-Fe2O3 was observed as 6.1, 6.9, and 7.0 nm. The smaller pore size and the larger surface area of the prepared nanoparticles can be dedicated to different electrochemical and biocompatibility applications.Furthermore, the surface area characteristics of the prepared samples were evaluated by the Brunauer–Emmett–Teller (BET) theory (BET) surface area analyzer. 3O4, carbon-modified Fe3O4 and α-Fe2O3 with a scan rate of 50 mV/s is shown in 3O4 was high compared to α-Fe2O3, which could also be the reason for the larger integrated CV curve area for Fe3O4 compared to α-Fe2O3.The cyclic voltammogram of FeCs) of the carbon decorated Fe3O4 and α-Fe2O3 electrode materials was calculated from the CV curves using the equation∫idV is the integrated area of CV curves, m is the mass of electrode material on the electrode (g), ΔV is the potential window range, and v is the scan rate (V/s). The specific capacitance of the carbon decorated Fe3O4 and α-Fe2O3 was calculated as 292 and 290 F/g, which was higher than 198 F/g of bare Fe3O4. The enhanced capacitance value was observed around 54% for carbon-modified samples, which is the result of carbon addition within the composite materials. The linear decreasing order of specific capacitance rate was observed with increasing scan, which might be due to the internal resistance within the electrode, limiting the charge transfer process  curve of carbon-modified α-Fe2O3 with different current densities ranging from 0.5 to 20 A/g was recorded and shown in I (amp) is the applied current to the electrode, t is time taken for discharging (s), m is the mass of active electrode material (g), and ΔV is the potential window.Furthermore, galvanostatic charge/discharge studies are carried out to confirm the capacitive nature of Fe3O4, carbon-modified Fe3O4, and α-Fe2O3 is shown in 3O4. The carbon-modified α-Fe2O3 showed a specific capacitance of 192 F/g with a current density of 1 A/g, which was higher than that of bare Fe3O4 of 147 F/g. This enhancement may have originated from the higher BET-specific surface area and increased pore size of carbon-coated Fe3O4, which increases the electrolyte/electrode contact area and hence provides a greater number of active sites to store the charges. The larger value of specific capacitance with the modification carbon on Fe3O4 corroborated with the CV results. A decreasing trend in specific capacitance was observed with increasing current densities. At low current densities, a greater number of charges accumulated in the inner active sites of the electrode result in high specific capacitance values.The calculated specific capacitance from the galvanostatic charge/discharge curves for bare Fe3O4 composite electrodes was carried out to verify our previous results and their corresponding Nyquist plots are illustrated in 3O4. The data were evident to the fact that the introduction of carbon on Fe3O4 has significantly reduced the charge transfer resistance. Moreover, all three spectra showed straight line shape at low frequency, which is regarded as an important feature of a capacitor. Among the different spectra, F2 electrode showed the steeper line than other electrodes, the vertical shape of the curve confirmed its good capacitive performance.Besides, EIS of Fe3O4 showed slightly lower than bare Fe3O4, which is favorable for electron transport. 3O4, carbon supported Fe3O4, and α-Fe2O3 samples for varying frequencies. The phase angle of all samples approach to zero at high frequency, but the phase angle of each electrode is distinguishable in the lower frequency region (less than 10 Hz). The phase angle of bare and carbon supported composites are very closer to −90°, which confirms the ideal capacitive nature of the prepared electrodes. Furthermore, to evaluate the cyclic stability of the electrode, all the samples were tested for 7,000 cycles at high current density of 5 A/g , proline (pro), methionine (Met), glycine (Gly), and valine  through hydrogen bonding with binding affinity of −11.5 kcal/mol; these interactions can be attributed to the influential change in structural and functional integrity of sod1 [3O4 nanoparticle can be seen in The observation can be attributed toward the lower internalization of α-Fee stress . Hence, enomenon . The fac of sod1 . The var2O3/C nanoparticles compared to Fe3O4 nanoparticles will lead toward less apoptosis. The hypothesis was experimentally verified by Acridine orange staining, which has been established as a known marker dye to determine the cellular apoptosis in cells [2O3/C nanoparticles exposed to embryos. The data were in correlation with our assumption and evident to the verification of hypothesized fact. Similar observation has been found by different literature in case of exposure to other metallic oxide nanoparticles like ZnO, CuO, and TiO2 nanoparticles [in vivo biocompatibility of α-Fe2O3/C nanoparticles compared to bare Fe3O4 nanoparticles can be deduced as follows: carbon modification of Fe3O4 nanoparticles induce higher surface area with higher porosity and less magnetization saturation. The alteration of these parameters causes less attachment and internalization of α-Fe2O3/C nanoparticles at the surface and inside the embryos. Hence, the induction of hypoxic condition is comparatively less leading to less generation of ROS. Moreover, due to less internalization that occurred as a result of less magnetization value, the interaction of α-Fe2O3/C nanoparticles would have been less with protein molecules inside the cells which are responsible for cellular physiology like oxidative stress and apoptosis. The combinatorial effect of these two phenomena leads toward the less oxidative stress and less apoptosis in case of α-Fe2O3/C nanoparticle exposure compared to Fe3O4 nanoparticles.Dysregulation of oxidative stress has been reported to be one of the major cause of cellular physiology abnormalities and enhancement in cell death activities like apoptosis . Hence, in cells ,42. As sarticles ,43,44. Tarticles  and our 3O4 nanoparticles with novel method and indicated toward the higher biocompatibility.Hence, the detailed study evident toward the higher utility of synthesized carbon-modified Fe43O4 by simple chemical oxidation method. The chemically prepared MNPs were surface modified with carbon by chemical pyrolysis process. The single phase of bare Fe3O4 and phase change due to higher calcination temperature was confirmed from XRD analysis. The octahedral-shaped morphology and the particle size distribution were analyzed by FESEM, TEM, and HRTEM microscopic measurements. Bare and carbon-modified Fe3O4 showed the highest saturation magnetization of 90 and 82 emu/g. α-Fe2O3/C sample exhibited the lesser saturation magnetization of 24 emu/g. The reduction in the saturation magnetization is due to the non-magnetic fraction of carbon materials and the phase transformation occurred at higher calcination temperature. The capacitive behavior of the prepared samples was analyzed by cyclic voltammogram and galvanostatic charge/discharge studies. The highest specific capacitance of 213 F/g was observed for carbon-modified Fe3O4 and carbon-modified α-Fe2O3 showed a specific capacitance of 192 F/g. The electrochemical studies also confirmed that incorporation of carbon with Fe3O4 significantly reduced the charge transfer resistance and increased the capacitive nature. Cyclic performance was tested up to 7,000 cycles. In vivo biocompatibility analysis showed reduction in toxicity of Fe3O4 nanoparticles by carbon modification by inducing less morphological abnormalities, oxidative stress, and apoptosis in zebrafish embryos. The higher magnetization, superior electrochemical characteristics, and higher biocompatibility suggested that the carbon-modified Fe3O4 would be the best among other two materials which are suitable for the negative electrode materials for supercapacitor applications and will be also ecofriendly with respect to environmental and clinical aspects.We successfully synthesized novel octahedral-shaped morphology of FeSuresh K. Verma: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Arun Thirumurugan: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization. Pritam Kumar Panda: Conceptualization, Investigation, Software, Methodology. Paritosh Patel: Methodology, Formal analysis, Investigation, Resources. Aditya Nandi: Methodology, Investigation, Formal analysis. Ealisha Jha: Methodology, Formal analysis, Investigation, Resources. K. Prabakaran: Methodology, Formal analysis, Investigation, Resources. R. Udayabhaskar: Methodology, Formal analysis, Investigation, Resources. R.V. Mangalaraja: Methodology, Formal analysis, Investigation, Resources. Yogendra Kumar Mishra: Methodology, Formal analysis, Investigation, Writing – review & editing, Resources, Formal analysis. Ali Akbari-Fakhrabadi: Methodology, Formal analysis, Investigation Writing – review & editing, Resources. Mauricio J. Morel: Methodology, Formal analysis, Investigation Writing – review & editing, Resources. Mrutyunjay Suar: Conceptualization, Methodology, Software, Validation, Resources, Data curation, Writing – review & editing, Supervision, Project administration, Funding acquisition. Rajeev Ahuja: Conceptualization, Methodology, Software, Validation, Resources, Data curation, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Our calculations at various levels of theory suggested that the Te···Te chalcogen bonding in (WTe2)2 is weak, whereas the Se···Se and S···S bonding interactions in (WSe2)2 and (WS2)2, respectively, are of the van der Waals type. The presence and character of Ch···Ch chalcogen bonding interactions in the dimers of (WCh2)2 are examined with a number of theoretical approaches and discussed, including charge-density-based approaches, such as the quantum theory of atoms in molecules, interaction region indicator, independent gradient model, and reduced density gradient non-covalent index approaches. The charge-density-based topological features are shown to be concordant with the results that originate from the extrema of potential on the electrostatic surfaces of WCh2 monomers. A natural bond orbital analysis has enabled us to suggest a number of weak hyperconjugative charge transfer interactions between the interacting monomers that are responsible for the geometry of the (WCh2)2 dimers at equilibrium. In addition to other features, we demonstrate that there is no so-called van der Waals gap between the monolayers in two-dimensional layered transition metal tungsten dichalcogenides, which are gapless, and that the (WCh2)2 dimers may be prototypes for a basic understanding of the physical chemistry of the chemical bonding environments associated with the local interfacial/interlayer regions in layered 2H-WCh2 nanoscale systems.Layered two-dimensional transition metal dichalcogenides and their heterostructures are of current interest, owing to the diversity of their applications in many areas of materials nanoscience and technologies. With this in mind, we have examined the three molecular dimers of the tungsten dichalcogenide series, (WCh D2, ,12, wherivative) ,14,15, aivative) ,17.2 structures, each B–A–B monolayer comprises three atomic layers, with the transition metal (A) layer sandwiched between two chalcogen layers  [2 systems [Although several experimental and theoretical studies have been reported on ABsystems) ,34,35,36systems) . In the systems) ,22,38,39 systems . These a2 systems [The importance of transition metal dichalcogenide materials, and what appears to be an incomplete understanding of the nature of the chemical bonding in the interfacial region between the monolayers of the metal dichalcogenide bilayer and multi-layered systems, prompted this study. As in our recent report on MoCh systems , we focu systems , with an systems ).2 , are discussed.  [The geometric, electronic, orbital, energetic, and charge-density based bond critical point and isosurface topologies of bonding interactions responsible for the formation of the three dimers, WChng (ChB) .) The orng (ChB) ,46, wherng (ChB) , interacng (ChB) , indepenng (ChB) ,50, and ng (ChB)  approachng (ChB) ,53,54, i2)2, and those known for the crystalline phase, are compared to address the titled concern: whether or not the chalcogen-centered non-covalent bonding interactions in (WCh2)2 dimers can serve as prototypes for a fundamental understanding of the local interfacial/interlayer chemical bonding environment in two-dimensional, layered tungsten dichalcogenides.The intermolecular geometries that emerge from the gas phase calculations on  between the tungsten bonded Ch sites in the WS2 and WSe2 are less than twice the vdW radius, 3.60 and 3.80 Å, of the S and Se atoms, respectively, whereas that of 4.007 Å in WTe2 is slightly longer than twice the vdW radius of Te, 4.00 Å (Bondi’s radius used: rvdW (S) = 1.80 Å; rvdW (S) = 1.90 Å; rvdW (S) = 2.00 Å) [2 are weaker than the S···S and Se···Se bonds in WS2 and WSe2, respectively, and are of van der Waals type. Whether or not this immediate conclusion is misleading can only be verified once the energy of these interactions is calculated. We discuss this in a following section that summarizes the binding energy of the WCh2 dimers.The S···S, Se···Se, and Te···Te inter-layer distances in the respective systems are either less than (for the former two) or slightly greater than twice the van der Waals radius of the respective chalcogen atom. For instance, the interlayer bond distances of 3.357 and 3.738 Å in WS 2.00 Å) ). From t2 crystals are significantly bent, with ∠W–Ch···Ch = 140.2°, 139.6°, and 140.3° when Ch = S, Se, and Te, respectively. The non-linearity in these interactions could be a consequence of a relatively less negative Ch site in one monolayer interacting attractively with a relatively more negative junction region, delocalized over the triangular face formed by the three Ch sites, on the neighboring layer. This becomes evident if one examines the distance between the Ch site and the centroid of the triangular Ch···Ch···Ch face in the second monolayer. This distance is 2.804, 3.110, and 3.463 Å in WS2, WSe2, and WTe2, respectively . This overlapping is readily appreciated when one relates the Ch···Ch interlayer bond distance with the sum of the van der Waals radii of interacting Ch atomic domains. The Bondi’s van der Waals radii of S, Se, and Te are 1.80, 1.90, and 2.0 Å, respectively [2, WSe2, and WTe2 crystals, respectively. Since the van der Waals radii for all atomic domains of the periodic table are associated with an error of approximately 0.2 Å [2 systems.The question that arises then is where is the van der Waals gap that is often referred to as existing between the monolayers in a bilayer 2H system of the WChectively . Therefoly 0.2 Å , it seem2, the interlayer Ch···Ch bond distance in the crystal geometry is 4.007 Å and is within the radii error limit of twice the van der Waals radius of Te. This suggests that there is no van der Waals gap between the layers in either of the three crystal systems, as the Ch sites of the two interacting monolayers are bonded with each other through weak attractive forces (vide infra). That there is no physical gap (only voids) between the monolayers of WCh2 is also evident from the space-filling model shown in In the case of WTeS,minV and S,maxV, the local most minimum and local most maximum of potential, respectively. Each can be positive or negative depending on the nature of the electron density depletion or accumulation at specific regions on the molecular surface [S,maxV, on the surface of a bonded atom is commonly observed on the outer extension of a covalent bond, and the local most minimum of potential, S,minV, is commonly observed in a region dominated by π-density and lone-pair electron density [The MESP model  has been surface ,67,68. W surface ,69,70. T density ,69 . The strength of the σd hole on W is very similar in the three monomers: 39.8 kcal mol−1 in WS2, 39.5 kcal mol−1 in WSe2 and 39.6 kcal mol−1 in WTe2, which explains why W accepts multiple chalcogen-centered bonds in the WCh2 crystals , which is not unexpected given that the metal center has empty, electron-deficient dtals see . This altals see . σp); they decrease in magnitude in the order WTe2  > WSe2  > WS2 , a trend that is consistent with the calculated binding energy of the WCh2 dimers (vide infra).By contrast, the axial portions of the Ch atoms bonded to tungsten are relatively more positive than the lateral portions that appear, firstly, along, and, secondly, around the W–Ch bond extensions. The first are nothing other than p-type σ-holes  of QTAIM, since the latter is homeomorphic to the former, with few exceptions. While the LOL analysis encloses a large number of critical points, we restricted ourselves to the  attractors in the gradient vector field of LOL. As Jacobsen noted previously [We carried out an analysis of the electron localization function (ELF)  and the  systems ,78,79. A systems  that fol systems ,73, a coeviously , the crieviously . LOL alseviously .2 is characterized by one  attractor, which represents locally maximal electron localization, for which, the ELF and LOL values corresponding to this attractor are 0.766 and 0.626, respectively. In addition, each Ch atom of the monomer features a single attractor expected of the valence lone-pair, with ELF and LOL values of 0.889 and 0.621, respectively, in the case of WS2. The tungsten center in the monomers is surrounded with a quintet of lone-pair attractors; two of them are along the outer extension of the two Ch–W axes . For WSe2, the ELF (and LOL) values are 0.688 (0.581), 0.811 (0.494), and 0.888 (0.735) for the  attractors responsible for the W–Se bonds, Se’s lone-pair, and W’s lone-pair along the Se–W bond extensions, respectively. For WTe2, these values are 0.645 (0.556), 0.748 (0.441), and 0.887 (0.734), respectively. Other than the valence shell lone-pair attractor around the Ch atom noted above, there are two other lone-pair attractors located in close vicinity to each Ch atom in WCh2 molecules. This suggests that the ELF and LOL models are superior to the MESP model in identifying the detailed topology of bonding and non-bonding pairs in this type of molecule.Our calculations indicate that each W–S bond in WS2 have a multiple bond character (vide infra), the LOL picture still shows one attractor for each bond in the monomer. This mirrors the results in the study of Jacobsen, who found a single attractor for both C–C and C=C bonds, with a larger LOL for the former of 0.822 compared to 0.776; a value of 0.721 was found for the triple bond in N2 [2H6), 0.9412 (for C=C double bond in C2H4), and 0.8906 (for C≡C triple bond in C2H2) have been reported [2 series, where the LOL between the bonded atomic basins systematically decreased as one passes from WS2 through WSe2 to WTe2.While the W–Ch bonds in WChnd in N2 . When acreported ,81. This2 monomers are shown in The 2D maps of the ELF and LOL functions for the molecular plane defined by the two Ch atoms and one W atom for the three WCh2)2  dimers obtained with M06-2X/aug-cc-pVTZ(-PP) are shown in 2)2  dimers, the Ch···Ch bond distances increased marginally by 0.003 Å, whereas, for (WTe2)2, the Te···Te bond distance decreased by 0.001 Å. The ∠W1–Ch2···Ch6 and ∠W4–Ch6···Ch2 angles increased by 0.2° for the first two dimers and remained unchanged for the latter. The ∆E and ∆E(BSSE) both increased by 0.1 kcal mol−1, yet no change was noticeable in the dipole moments of all three dimers. This indicates that the effect of dispersion on the dimer properties is very marginal when it is incorporated with the M06-2X functional.The geometrically relaxed (WChr(Ch···Ch) and ∠W-Ch···Ch for all three (WCh2)2 dimers.Among the three basis sets tested, the best Ch···Ch intermolecular bond distances  values in E(BSSE) is markedly smaller than ∆E for each of the three dimers with M06-2X/LANL08 and M06-2X-D3/LANL08. Nevertheless, the ∆E(BSSE) values obtained with the highest basis set applied suggest that the Ch···Ch interactions in the three (WCh2)2 dimers are of van der Waals type, since interaction energies of van der Waals complexes are generally close to, or below, −1.0 kcal mol−1, and are smaller than weak interactions in weakly bound complexes  [An inspection of the ∆l mol−1) ,83,84.To determine the validity of this conclusion, we computed the binding energies of all three dimers using MP2(full), B97-D3(BJ), PW6B95-D3(BJ), and B3LYP-D3(BJ) using the M06-2X/Aug-cc-pVTZ(-PP) computed equilibrium geometries. The results are compared with E and ∆E(BSSE) obtained with all of these four methods are larger than those computed with M06-2X and M06-2X-D3, suggesting that dispersion indeed plays an important role in elevating the complexation energies of the three dimers. The BSSE in energy is calculated to be very large with the post-Hartree–Fock method, MP2(full), and the trend in ∆E and ∆E(BSSE) across the dimer series is not systematic. That is not the case with all the DFT functionals used, which give a systematic increase in the bonding energy when passing from (WS2)2 to (WSe2)2 to (WTe2)2. This is also observed for ∆E(BSSE) as expected, given that Te is significantly more polarizable and less electronegative than Se, which, in turn, is more polarizable and less electronegative than S.The values of ∆E(BSSE) close to, or larger than, −2.0 kcal mol−1 for (WTe2)2, showing that this dimer is not a van der Waals system. That may also be the case with (WSe2)2, since ∆E(BSSE) gives the predicted values of −1.16, −1.48, and −1.73 kcal mol−1 with PW6B95-D3(BJ), B97-D3(BJ), and B3LYP-D3(BJ), respectively. (MP2(full) predicts a spurious ∆E(BSSE) of −3.66 kcal mol−1, larger than that of (WTe2)2). By contrast, and despite the S···S intermolecular distance in (WS2)2 being significantly smaller than the Se···Se and Te···Te in (WSe2)2 and (WTe2)2, respectively (see 2)2 is a van der Waals system.With the exception of M06-2X and M06-2X-D3, all other computational methods employed give ∆2)2 dimer can be rationalized from the molecular graphs shown in The QTAIM description of bonding interactions present in each 2 and largest, 0.0077 a.u., for (WTe2)2. Interestingly, the trend in the ρb values is concordant with the BSSE-corrected binding energies calculated for the three (WCh2)2 dimers (see E(BSSE)), suggesting that the charge density at the Ch···Ch bcp is indeed a measure of its bond strength.The charge density ρb at a Ch···Ch bcp is significantly smaller than that at a W–Ch bcp; the first are due to weak interactions between Ch atoms and the latter are the consequence of ionic bonds with some covalency. The small but non-zero values of ρb at the bcp of the Ch···Ch bonds are an indication of a bonded interaction signaled by QTAIM. The MESP model does not provide a way of quantifying the interaction. It does, however, enable one to see whether there is an atom–atom overlapping in the intermolecular region. This is indeed evident in 2)2 dimers are linked with each other.Obviously, ρ = 0.001 a.u. density envelope. The closest distance between a nucleus in a molecular entity and its van der Waals surface is called a “non-bonded atomic radius” [2)2, (WSe2)2, and (WTe2)2, the non-bonded radii of S2/S6, Se2/Se6, and Te2/Te6 atomic domains (see 2)2, (WSe2)2, and (WTe2)2 dimers, respectively, suggesting that the mutual penetration between the two Ch sites responsible for the intermolecular region is strongest in (WTe2)2. Since the larger the mutual penetration distance between bonded atomic basins, the stronger the interaction between them, this accords with the trend observed in the binding energy of the dimers  or open-shell (∇2ρb < 0) type [The sign of the Laplacian of the charge density, ∇ 0) type ,93. A cl 0) type —would be2ρb > 0 at the Ch···Ch bcps of the (WCh2)2 dimers, with the largest value of 0.0195 a.u. for (WS2)2 and the smallest, +0.0169 a.u., (WTe2)2. ∇2ρb = −0.0769 a.u. at the W–Ch bcps of the (WS2)2 dimer, whereas ∇2ρb > 0 at the W–Ch bcps for the (WCh2)2  dimers. This suggests that the W–S bonds in (WS2)2 are significantly more covalent than the W–Ch bonds in (WCh2)2 . That polar covalent bonds have ∇2ρb > 0 is not surprising [2 crystals [As expected, we found ∇rprising , and ourcrystals .Hb, which is the sum of the potential and gradient kinetic energy densities, Vb and Gb, at the bcps. In QTAIM, when the potential energy density significantly dominates over the gradient kinetic energy density at the bond bcp, then the Hb < 0, which is indicative of an open-shell interaction. When the gradient kinetic energy density dominates at the bcp, Hb > 0, the signature of a closed-shell interaction is revealed.This conclusion above can be verified by considering the sign and magnitude of the total energy density, Hb > 0 for the Ch···Ch interactions in the three (WCh2)2 dimers; they are of the closed-shell type and can therefore be regarded as non-covalent interactions. On the other hand, the W–Ch bonds in the three dimers have Hb < 0, and become less negative in the order (WS2)2 > (WSe2)2 > (WTe2)2. These results are suggestive of W–Ch ionic bonds with a distinctive covalent character. Indeed, the combined signature ∇2ρb > 0 and Hb < 0 suggest a mixed ionic and covalent character that increases with a decrease in the electronegativity, and an increase in the polarizability of the chalcogen across the series from S through Se to Te.Our results show that 2 unit responsible for the dimer is negative; the charge on each of S, Se, and Te are −0.388, −0.240, and −0.075 e for (WS2)2, (WSe2)2, and (WTe2)2, respectively, with a decrease in negative charge paralleling the decrease in the electronegativity of the chalcogen. The corresponding charge on W is 0.782, 0.483, and 0.147 e, respectively. These are somewhat different from the values found for the same atoms in the isolated monomers . Clearly, the dimer formation is accompanied by a rearrangement of charges, showing an effect of electrostatic polarization when the two monomers interact to form a dimer. Note that the magnitude of charges on the W and Ch atoms in the isolated monomers are substantially decreased compared to the formal charges +4.0 and −2.0 of W and Ch ions, respectively, showing that W becomes increasingly less positive, and Ch becomes increasing more positive. This implies that there is a significant transfer of charge from the W4+ ions to the Ch2− ions during the formation of the isolated monomers. The atomic charge is an informative quantum mechanical property of atoms in molecules  and crys2 crystals have significant covalent character, and a degree of polarity in the bond is caused by charge transfer from the metal to the chalcogen atoms [2, the electronegativity difference between W and Ch is 0.28, 0.10, and 0.07 for S, Se, and Te-containing systems, respectively; on this basis alone, one would conclude that the W–Ch bond is still a polar covalent bond, as has been deduced by others [The rearrangement of charge under the circumstances given above has been noted in numerous occasions previously. For example, it was argued that M–Ch bonds in Mo-Chen atoms ,99,100. y others ,102. Alty others ,104,105.μ. Our M06-2X/aug-cc-pVTZ(-PP) level calculation gave dipole moments of 2.73, 2.41, and 2.09 D for the monomers WS2, WSe2, and WTe2, respectively. Dimerization resulted in dipole moments of 5.30, 4.69, and 4.09 D for (WS2)2, (WSe2)2, and (WTe2)2, respectively. There is a clear enhancement of the dipole moment on the formation of the dimer, suggesting an increasing polarity of the dimers at equilibrium. A judgement on the polarity of a molecular domain can be made based on its dipole moment, g, DORI, and the recently proposed IRI-based NCI approach. Each has its limitations, and hence a joint application of these approaches to chemical systems may lead to a deeper understanding of the bonding involved between interacting atomic domains.A number of charge-density-based approaches have been developed within the last decade and applied to many chemical systems to explore chemical bonding interactions ,106,107.λ2) × ρ vs. RDG domains, where λ2 (λ1 ≤ λ2 ≤ λ3) is the second eigenvalue of the Hessian of the charge density matrix, and ρ is the charge density. RDG describes the reduced charge density gradient around the bond critical point region; the lower bound of RDG is zero and is reached whenever the charge density gradient vanishes at the bcp, a dimensionless quantity within the generalized gradient approximation of the exchange correlation term in DFT Hamiltonians [The RDG-based NCI characterization involves the plot of the sign and/or repulsive (λ2 > 0) interactions, and ρ quantifies the strength of that interaction. When the quantity sign(λ2) × ρ is mapped onto the RDG isosurface, both the nature and strength of the interactions within molecular entities become evident [The sign of al point . Clearly evident .λ2) × ρ vs. RDG plots are shown in 2)2, (WSe2)2, and (WTe2)2, respectively. The circular isosurface volume appearing between two Ch atoms (Ch6 and Ch2) in these dimers clearly corresponds to low-density regions, as shown by the greenish spikes in the sign(λ2) × ρ vs. RDG plots in the region −0.01 a.u. < sign(λ2) × ρ < 0.0 a.u. In this region, the sign(λ2) is negative. These are the characteristics of non-covalently bonded Ch···Ch interactions in all three dimers. There is a second spike in each of the three sign(λ2) × ρ vs. RDG plots, where sign(λ2) is positive. Since these appear in the low-density regions, with 0.0 a.u. < sign(λ2) × ρ < 0.01 a.u., these are signatures of van der Waals interactions.The sign × ρ vs. IGM(δginterintra/) for the three dimers. The spikes colored black are due to intramolecular interactions (W–Ch bonds), and those colored red are intermolecular interactions. The first appear in the high-density regions, and the second in the low-density regions. The IGM-based isosurface volumes that show up between the Ch atoms in (WS2)2, (WSe2)2, and (WTe2)2 correspond to the weak red spikes, and the strength of the interaction increases in this order: (WS2)2 < (WSe2)2 < (WTe2)2.A descriptor of the model, δλ2) × ρ vs. IRI for the dimers (WS2)2 and (WSe2)2, respectively, whereas that of the dimer (WTe2)2 is shown in g models, respectively, as each model requires a specific range of isovalues to generate isosurfaces of interest. The blue isosurfaces signify a significant accumulation of the charge density in the bonding region between W and Ch atomic basins, as expected for ionic bonding with some covalent character. The green isosurfaces indicate low-density regions, as similarly observed in the RDG and IGM-δg plots that appear between the non-covalently bonded Ch atoms. The spikes in the sign(λ2) × ρ vs. IRI plots corresponding to these isosurfaces are identical to those of the sign(λ2) × ρ vs. IGM(δginterintra/) plots, indicating that both models provide a similar insight into the atomic basins causing the interactions and their strengths.The more recently proposed IRI model  has an aIBSI) that emerges from the IGM formulation [pair signature, and, hence, the IBSI index. This means that the IGM and its δg descriptor are a way forward to locally quantify the electron density interpenetration from wavefunction calculations. These workers correlated the IBSI index with a number of conventional bond orders , albeit with poor regression coefficients, and called IBSI a new complementary index that is related to the bond strength. Considering A (as W1Ch3Ch2) and B (as W4Ch5Ch6) as two fragments of the (WCh2)2 dimer system 2, (WSe2)2, and (WTe2)2, respectively, which are the pair of atoms that are closest to each other. Accordingly, the pair Ch2···Ch6 have the largest δg indices of 0.135, 0.163, and 0.209 a.u. for the corresponding systems, respectively, suggesting that the two Te atoms contribute largely to the Te···Te interaction in (WTe2)2 compared to that contributed by the two S and two Se atoms for the S···S and Se···Se interactions in (WS2)2 and (WSe2)2, respectively. Similarly, the sum of the related IBSIW (IBSI for weak interaction) for Ch2/Ch6 was 1.114, 1.258, and 1.413 a.u./Å2, respectively. The IBSIW indices for the Ch2···Ch6 pair were 1.034, 1.179, and 1.349 a.u./Å2, respectively, where IBSIW for any atom–atom pair i and j is defined by Equation (1) and Equation (2).i,jd is the distance between atoms i and j in Å.Our calculations gave IBSIW index, the stronger the interaction, it is clear that the preference of stability follows the order of (WS2)2 < (WSe2)2 < (WTe2)2, and is in accordance with the trend in the observed binding energies of these dimers 2 dimers. The dominant delocalizations involve BD(3)W1–Ch2→BD*(3)W4–Ch6, BD(3)W4–Ch6→BD*(3)W1–Ch2, LP(2)Ch2/LP(2)Ch6→BD*(3)W4–Ch6/ BD*(3)W1–Ch2, where LP(2), BD(3), and BD*(3) represent the second π-type lone-pair, dπ-type bonding, and anti-bonding-type orbitals, respectively. To give an example, the second order charge transfer delocalization energies E(2) for these interactions were found to be 0.8, 0.8, and 0.5 kcal mol−1 for (WTe2)2, respectively. These results suggest that there are mutual hyperconjugative charge transfer interactions between the two monomers when they form a dimer. That W–Ch bonds in isolated WCh2 and complexed (WCh2)2 have the triple bond character, which was involved in the charge transfer delocalization, was evidenced by a bond order analysis [2)2, respectively. Very similar results were obtained for the W–Se and Se···Se interactions in (WSe2)2 and W–S and S···S interactions in (WTe2)2. The bond orders suggest that the link between W and Ch is of a triple-bond character, whereas that between the two Ch atoms (Ch···Ch) of the two interacting monomers forming the dimers is of a single-bond character. We note that the Mayer bond order [Finally, we note that the attractive interaction between the WChanalysis ,113,114.nd order  essentiand order ,111,116)2 monomers and the three (WCh2)2 dimers were examined in this study using the DFT-M06-2X functional. The construction of the dimers was made possible by inspecting the geometry of the interfacial region between the WCh2 layers, reported in the crystals of WCh2 in the solid state [Three V-shaped WChid state ,117,118.2 crystals, we did not explore any other dimers in the conformational space. However, we are interested in exploring this conformational space to identify the global minimum for each dimer, and then to examine the extent to which the nature of the bonding environment and bonding energies between the two monomers changes upon dimerization; we will discuss this elsewhere. Due to the fact that we were specifically interested in a basic understanding of the nature of the interactions between Ch sites in the interfacial/interlayer region of WCh2 crystals. This phase is semiconducting, and stable at room temperature. Reports show that another phase, the 1T phase, is highly unstable and converts back into the 2H phase with time and a variation in temperature. Metastable 1T(1T′) phases require higher formation energy compared to the thermodynamically stable 2H phase; thus, in standard chemical vapor deposition and vapor transport processes, the transition metal dichalcogenide materials normally grow in the 2H phases [2 is shown in We have also limited our study to providing insight into the nature of the interfacial bonding in the 2H phase of the WChH phases ,119. The2 monomers and (WCh2)2 dimers. We show that this functional reproduces the geometry of the intermolecular interactions found in the interfacial regions in the crystalline phase ), for the (WCh2)2 dimers was calculated using the supermolecular procedure proposed by Pople [E was obtained by subtracting the sum of the total electronic energies (ETsum(monomers)) of the respective isolated monomers forming the dimers from the total electronic energy of each dimer (ET(dimer)). The energies of the monomers, as in the fully relaxed dimer configurations, were utilized. The basis set superposition error (BSSE) was calculated using the counterpoise procedure of Boys and Bernardi [E(BSSE) is the BSSE energy.The binding energy, ∆by Pople . ∆E was Bernardi , EquatioE of all three dimers using M06-2X-D3, MP2(full), B97-D3(BJ) [For reasons given in a following section, and because they are useful for the study of non-covalent interactions ,128, we 7-D3(BJ) , PW6B95-7-D3(BJ) ,130, and7-D3(BJ) ,132, whe7-D3(BJ) , and D3, the bond path, and the molecular graphs) were obtained using the AIMAll software [2)2 dimers were obtained using MultiWfn and VMD software [The topological properties 2  dimers examined at the M06-2X level of theory, in conjunction with three different basis sets: LANL08, def2-TZVPPD, and Aug-cc-pVTZ(-PP). We showed that, though each basis set has its own limitations, they all provided a very consistent Ch···Ch intermolecular bond distance for the three dimers examined and are well comparable with the interfacial Ch···Ch geometries observed for the WCh2 crystals in the room temperature 2H-phase. The overall performance in obtaining intermolecular bond distances and angles of approach leading to the formation of the three dimers is shown to be described well with the Dunning’s pseudopotential basis set Aug-cc-PVTZ(-PP). Although the M06-2X functional provided an intermolecular geometry in reasonable agreement with the experimentally established solid state geometry of WCh2, it underestimated the binding energies of the dimers. The effect of Grimme’s dispersion with the original D3 damping function had a negligible effect on the geometric, electronic, and energetic properties of the dimers, especially when it was incorporated with the M06-2X functional. However, the uncorrected and BSSE-corrected binding energies calculated on the M062X/Aug-cc-pVTZ(-PP) geometries of the dimers with MP2(full), B97-D3(BJ), PW6B95-D3(BJ), and B3LYP-D3(BJ) enabled us to demonstrate that (WSe2)2 and (WTe2)2 are weakly bonded dimers, and that (WS2)2 is a van der Waals system. That the (WS2)2 is a van der Waals system is a result of the attraction between the W-bound S atoms with positive electrostatic potentials along the W–S bond extensions in the isolated WS2 monomers, as has been observed for other systems before [In this study, we discussed the geometric, electronic, charge density topology, and energetic properties of 2 dimers. The origin of the Ch···Ch interactions in (WCh2)2 was revealed by the second order hyperconjugative estimates of donor acceptor interactions in an NBO analysis, which showed that there are several weak charge transfer delocalizations between the interacting monomers at the dimer geometry, including, for example, LP(Ch)→BD*(3)W–Ch and BD(2/3)W–Ch→BD*(3)W–Ch. We therefore propose that the characteristics of the chalcogen–chalcogen bonding interactions observed in the (WCh2)2 dimers of tungsten sulfide, tungsten selenide, and tungsten telluride are prototypes for a basic understanding of the local interfacial/interlayer chemical bonding environment observed in the layered tungsten dichalcogenides in 2D, and that there is no van der Waals gap observed between the monolayers responsible for the interfacial/interlayer regions in bi-, tri-, and/or multi-layered tungsten dichalcogenides, but only voids.The QTAIM, IRI, IGM-δ