Nematodes represent >3/5 of the abundance of the world’s metazoans and usually account for nearly 90% of the total benthic fauna, playing a key ecological role in the benthic ecosystem functioning on a global scale. These small metazoans include a relevant number of microscopic predators and, in turn, are the most abundant preys of macro-megafauna and fish juveniles thus playing a key role in marine food webs. Here, using two independent approaches, we test the bioaccumulation in marine nematodes of several heavy metals present in contaminated sediments. We report here that nematodes, despite their short life cycle and small size, bioaccumulate significantly heavy metals. Bioaccumulation increases from deposit feeders and microalgal grazers to predators of microbes and other tiny metazoans. These results suggest that nematodes also contribute to their biomagnification along the food webs and can contribute to increase the transfer of contaminants from the sediments to larger organisms. Nematodes bioaccumulate contaminants and promote the channeling of heavy metals to higher trophic levels of the food chain. Predators of metazoans display the highest heavy metal concentrations, followed by predators of microbes and microalgal feeders. Nematodes include a large variety of feeding guilds (trophic groups), spanning from deposit feeders (that ingest organic detritus from the sediments) and microalgal grazers to predators of microbes (feeding on bacteria and protozoans) and predators of other nematodes and tiny metazoans3. In turn, nematodes are the preferred prey of larger organisms and bentho-nekton juveniles8. Thus, nematodes represent a crucial link between the benthic microbial food web and the higher trophic levels.Marine nematodes represent 2/3 of the metazoans on Earth. These small-sized organisms (<0.5 mm) are ubiquitous in all marine sediments from shallow waters down to hadal depths. Due to their high abundance, limited mobility, and short life span , nematodes significantly contribute to the biomass production in all marine ecosystems and dominate biomass production at abyssal depths9. Marine sediments also play a key role in the heavy metals diagenesis, and depending on the environmental conditions, can make xenobiotics more or less bioavailable to marine organisms10. Heavy metals bound to the sediment particles can be ingested by deposit-feeding organisms or can be absorbed/uptake directly by microbes and/or microalgae13. Heavy metals bioaccumulation  and biomagnification  can have severe biological consequences16.Marine sediments are a major reservoir of several pollutants, including heavy metals, as they adsorb on suspended particles, which are then transferred by sedimentation processes on the seafloor17, are permanently exposed to the contaminants present in the sediments and show a different degree of sensitivity to chemical pollution. When nematodes are preyed by fish juveniles and other predators, they can contribute to the transfer of xenobiotics to higher trophic levels. However, the nematode bioaccumulation and biomagnification of contaminants have been so far completely neglected, likely due to their short life span and tiny body size .Nematodes, due to their direct development and lack of planktonic larvae19, in their tissues. In addition, we explored the relationships between nematode body size and feeding type with their heavy metals’ bioaccumulation.To investigate the bioaccumulation and biomagnification potential in marine nematodes we assessed, using two independent methodologies, the content of seven heavy metals , some of which classified as priority pollutants in marine ecosystemsAll selected heavy metals, except for Ni, showed higher concentrations in chronically contaminated sediments of the Bagnoli-Coroglio Bay (hereafter “contaminated sediments”) than in the sediments of the Gabicce Mare in the Adriatic Sea (hereafter “control sediments”)  to 3.9 × 106 ind m−2 (St127) in contaminated sediments, while they ranged from 1.89 to 2.01 × 106 ind m−2 in the control sediments. Nematode assemblages in the contaminated sediments were dominated by microalgal grazers (2A) whose contribution to the total abundance ranged from 38 to 58% , followed by deposit feeders (1B) , predators of microbes (1A)  and predators of metazoans (2B) . In the control sediments, microalgal grazers represented the dominant group (50%), followed by deposit feeders (25%), predators of metazoans (19%) and predators of microbes (6%), respectively. The individual biomass of nematodes ranged from 0.03 to 0.41 µgC ind−1  to 378.6 mgC m−2 (St44) in the contaminated sediments, while values ranged from 120.0 to 127.6 mgC m−2 in the control site.Nematode abundances ranged from 0.65 × 10The results of the atomic absorption spectrophotometer analysis of As, Cd, Cr, Cu, Mn, Ni and Zn concentrations in the four nematode feeding types present in contaminated sediments are reported in Fig. We used the Log-Gamma model (GLM) on the content of heavy metals per feeding type, using the microalgal grazers (herbivores) as baseline of the food web, for testing differences in heavy metal bioaccumulations among feeding types and sediment contamination levels. The output of the GLM indicated that As, Cr, Mn and Ni bioaccumulation in predators of microbes 1A) and metazoans (2B) was significantly higher than in deposit feeders (1B) and microalgal grazers (2A)  for each feeding type estimated from the results of the X-ray microanalysis carried out on the tissues of nematodes from the contaminated and control sediments , while Zn was significantly higher in the head and middle body than tail, independently of the trophic groups in contaminated sediments  and heavy metal content in their tissues indicated the presence of a significant positive relationship only for Zn Fig. . Such re19. In large marine organisms, the concentration of heavy metals typically increases with their size/age (bioaccumulation process) and in higher trophic levels (biomagnification processes), thus it is expected that large, long-living organisms and top predators are the components most impacted by heavy metals22. Due to their short life span and small body size, nematodes are not expected to experience significant bioaccumulation or biomagnification of heavy metals14 and either soil and marine nematodes can tolerate high concentrations of heavy metals  in the sediments where they live25. Our results provide evidence of the bioaccumulation of heavy metals in marine nematodes. In fact, nematodes living in contaminated sediments contain in their tissue concentrations of As, Cd, and Cr significantly higher than those living in control sediments.Xenobiotics can exert major negative effects on marine biota, which depend upon the duration of exposure and concentrationOur estimates of heavy metal concentrations per individual biomass obtained from the X-ray microanalysis confirm that nematodes collected in the highly contaminated sediments display higher bioaccumulation of some heavy metals (Cr and Cu) compared to those from control site. This is also evident from the multivariate analyses carried out on the bioaccumulation of all heavy metals analysed, which clearly segregated the four nematode trophic groups of contaminated sediments from those of the control site Fig. .26. In our study, the bioconcentration factor changed among different trophic groups suggesting different responses of the nematode feeding types to the contaminants (see below). In particular, the bioconcentration factor for As, Cr and Ni was always significantly higher in the predators of metazoans (2B) and of microbes (1A) than in microalgal grazers (2A) and deposit feeders (1B), allowing us to hypothesize a transfer of these heavy metals from the preys (microbes and tiny metazoans) to the predatory nematodes. Different accumulation mechanisms of heavy metals can be hypothesized, including (a) passive diffusion through the body surfaces, (b) adsorption on the nematode cuticle; (c) direct ingestion of heavy metals adsorbed on organic particles or contained in preys27. Heavy metals accumulation in organism’s tissues may depend also on the metal bioavailability, which in the sediment is largely controlled by their repartition in different geochemical phases30. In this regard, previous studies carried out in contaminated sediments of Bagnoli-Coroglio Bay revealed that ca 6, 15, and 40% of the total concentrations of As, Cd and Mn, respectively, are present in the most dynamic and potentially bioavailable (exchangeable/carbonate) fraction for benthic organisms31.To assess the degree of “enrichment” of each heavy metal in the nematodes compared to its concentration in the sediments, we estimated the bioconcentration factor (based on the results of the atomic absorption spectrophotometry), although it should be used with caution as the values are inversely related to the metal concentration in the environmentCaenorhabditis elegans), which has been reported to have a limited adsorption capacity of chemical contaminants32. As reported for soil nematodes we hypothesize that also marine nematodes have a limited adsorption capacity of the heavy metals through the cuticle.The results obtained from the X-ray microanalysis also indicate that the relative heavy metal content is not homogeneous in all parts of the nematode body and that, among the investigated metals, Cu and Zn are generally higher in the middle part of the body (oesophagus) and in the head and lower in the posterior end/tail. These findings suggest that the accumulation of heavy metals in marine nematodes does not occur through the passive diffusion or adsorption on the nematode cuticle, rather it is due to the ingestion of food items. This conclusion is consistent with previous observations on the cuticle of a specific group of soil nematodes . However, given the complexity of the trophic pathways, specific laboratory experiments are needed to better understand the mechanisms of heavy metals incorporation in nematodes.Our study based on two independent approaches and multiple heavy metals analysis revealed that nematodes belonging to different trophic groups displayed significantly different concentrations of contaminants. Among the four trophic groups investigated, predators of other metazoans and microbes displayed the highest heavy metal concentrations when compared to microalgal grazers and deposit feeders. Biomagnification of As, Cr, Mn, and Ni was observed in predators of microbes and of other metazoans, while the biomagnification of Zn was more evident in predators of metazoans. The different levels of heavy metals in predators, and the lowest concentrations in microalgal grazers and deposit feeders suggest that nematodes can accumulate these contaminants differently because of their different diet and feeding behaviours. These findings provide evidence that these organisms, at the top of the benthic microbial food web, can magnify the contaminants through the food web . Previous studies reported that benthic diatoms can accumulate low amounts of heavy metals when compared to concentrations in the sediments15. Our results indicate that during their life, although some nematodes can be omnivorous or change their food targets, predators of metazoans (2B) and of microbes (1A) can bioaccumulate heavy metals in their body tissues more than microalgal grazers (2A) and deposit feeders (1B), resulting in potential biological effects in contaminated areas. These findings provide new insights to explain the varying tolerance of different nematode trophic groups to a mixture of heavy metals17 and suggest that nematodes can promote the channelling of different xenobiotics to higher trophic levels. This process has been completely overlooked so far but given the quantitative relevance of nematodes in all benthic food webs, we conclude that their contribution to xenobiotics’ bioaccumulation and biomagnification potential through the marine food webs should not be further neglected.Nematodes play a key ecological role in all marine benthic habitats and at all depths. They are the numerically dominant component of the benthic metazoan fauna , they feed on organic detritus, prokaryotes, protozoans, and other metazoans (as well as their carcasses) and thus represent a crucial food source for macrofauna and juvenile megafauna (including bentho-nekton components)46. In addition to the heavy metals derived from industrial activity , the site is exposed to a natural volcanic contamination of As, Cr, Ni, and V (geogenic forcing47). Sediment texture is characterized by coarse sand and sandy silt at shallower depths, and fine sands, silty and silty-clay sediments in the central part of the bay48. Heavy metals in the sediments can be also classified in term of their potential bioavailability for the nematodes as they are present in soluble forms or associated with Fe-Mn oxides, organic matter, sulphides, and carbonates31.The Bagnoli-Coroglio Bay  is a chronically contaminated area of the Mediterranean Sea. Here, the industrial activity, due to a steel plant using fossil coal, iron, and limestone, started in 1905 and ended in the ‘90 s. High concentrations of different metals  can be found from the surface down to >2 m depth within the sediments making this area among the most polluted of the Mediterranean Sea46 (Supplementary Table Sediment samples were collected using a Van Veen grab (25 L) in five stations: three stations located at depths ranging from 6 to 17-m, which were close to the industrial plant and characterized by the highest heavy-metal contamination, and two stations located at 8 and 14 m, respectively, in the adjacent areas characterized by a lower heavy metals contamination3, HF and H2O2 (EPA method 3052) and was analysed by inductively coupled plasma mass spectrometry  to detach organisms from the sediment particles. This treatment has proven not to cause any damage to the nematode cuticle−2 . Each body volume was multiplied by an average density of 1.13 g cm−3 to obtain the wet weight, which once converted into dry weight (µg dry weight: µg wet weight = 0.2557), was converted into biomass assuming a carbon content of 40% of the dry weight57.Finally, for each specimen, biomass was calculated from their biovolume, using the AndrassyTo assess the contamination in the nematodes of different trophic groups, we used two independent approaches: (1) the determination of the heavy metal concentrations based on the atomic absorption spectrophotometer analysis and (2) the analysis of the heavy metals composition using the quantitative X-ray microanalysis. This latter analysis was carried out to provide heavy metals composition in nematodes at individual level and in different body part of the animals, since current methodologies  do not allow the detection of heavy metal content in single specimens characterized by very low size.3  solution. Samples were then analysed using an Agilent DUO 240FS atomic absorption spectrometer  equipped with graphite furnace (GTA120 Graphite Tube Atomizer) and with Zeeman-effect background corrector. Argon (99.999% purity) was used as the carrier gas. Multi-element hollow cathode lamps were used as a light source. As, Cd, Cr, Cu, Mn, Ni and Zn were measured at wavelengths of 193.7, 228.8, 357.9, 324.8, 279.5, 232.0, and 213.9 nm, respectively. To improve the analytical measurements a 0.2% Pd matrix modifier in citric acid was used. Procedural blanks accounted for less than 1% of the total element concentrations in samples. The quantification of As, Cd, Cr, Cu, Mn, Ni and Zn was carried out using the calibration curve method (standard solutions of Titrisol grades from Merck) with at least three standard additions (and three measurements for each addition). The mean value obtained from each sampling area was validated with the method detection limits (LODs), calculated for each metal according to ICH Q2B: 0.08, 0.02, 0.15, 0.28, 0.06, 0.70, and 0.67 µg L−1, respectively, for As, Cd, Cr, Cu, Mn, Ni, and Zn58. To assess the accuracy of the data obtained from the instrumental analyses As, Cd, Cr, Cu, Mn, Ni and Zn were determined using BCR-414 certified reference material for plankton samples59. No statistically significant differences (P > 0.05) between certified and measured values were detected. Concentrations were reported as µg g−1.All analyses were carried out in three replicates for each of the four trophic groups  at the minimum accelerating voltage of 15 kV are: 5.411 for Cr, 8.040 for Cu, 6.398 for Fe, 5.894 for Mn, 7.471 for Ni and 8.630 for Zn. Nematodes, belonging to different genera/species divided by trophic group, were gradually dehydrated using different concentrations of ethanol, from 70% to 80%, 90%, 95% and 100% for one night. After critical point drying, nematodes samples were graphite-coated for the X-ray microanalysis. Using an analysis frame of 13 × 13 μm and a tension of 20kv, individuals belonging to each trophic group were analysed. Replicated analyses (n = 3) were conducted on (i) head, (ii) middle part of body and (iii) tail of each nematode genus/species to test potential differences in heavy metals composition among different tissues/organs/parts of the body. Different heavy metals, including essential  and nonessential/borderline (Cr) elements26 were identified. The content of each heavy metal was expressed as a percentage of their total content in the nematode body . Results of the X-ray microanalysis carried out on single nematode belonging to the four trophic groups were converted into heavy metal concentrations  based on the output of the atomic absorption spectrophotometer analysis to estimate metal content per individual biomass (pg µg-1).The X-ray microanalysis presents two major advantages for the determination of the elemental composition of an organism: (i) higher resolution (including single cells and subcellular components) and (ii) simultaneous identification of a wide range of elements. This approach has been already tested on meiofaunal organisms belonging to the taxon of Loricifera (length 400 µm and wide 90 µm) approximately of the similar size of the nematodes investigated in the present study61. Here, we used the following equation:nematodes is the heavy metal concentration determined through atomic absorption spectrophotometer analysis in the nematode tissues for each trophic guild and Csediments is the heavy metal concentration in the sediments. The concentration of heavy metals in nematodes and sediments was expressed as µg g-1.The bioconcentration factor (BCF) is used to evaluate the degree of enrichment of a heavy metal in an organism compared to that in its habitat0 represents the intercept term, and β1 and β2 are the coefficients corresponding to the factors Area and Trophic Group, respectively.A generalized linear model was used to describe the expected concentration of heavy metals in different nematode trophic groups from Bagnoli-Coroglio Bay and the control site and this model was fitted using maximum likelihood. This expresses the metal concentrations Y as35.Microalgal grazers were used as baseline since they feed on primary producers and are expected to bioaccumulate heavy metals to less extent compared with the other nematode feeding typesAnalyses were conducted using the GLM procedure in R.62. The similarity matrix in the heavy metals content of nematodes from Bagnoli-Coroglio Bay and the Gabicce Mare control site, based on the Bray–Curtis similarity (square root transformed), was applied to produce a non-metric multidimensional scaling two-dimensional plot (MDS).To visualize differences in heavy metals content among different trophic groups and areas, biplots after a Canonical Analysis of Principal Coordinates (CAP) were madep values were obtained from Monte Carlo sampling method63.PERMANOVA was applied to test for differences in the heavy metals composition  in different body parts comparing head, middle body, and tail in the four nematode trophic groups in Bagnoli-Coroglio Bay and in the Gabicce Mare control site using a three-way distance-based permutational analyses of variance based on unrestricted permutations of the raw data. All univariate analyses were carried out on Euclidean distance of untransformed data, using 999 permutations of the residuals under a reduced model. For all analyses, when significant differences were observed, pair-wise tests were also carried out. Because of the restricted number of unique permutations in the pair-wise tests, 63.PERMANOVA, CAP and MDS analyses were performed using the routines included in the PRIMER v7 and PERMANOVA softwareFinally, a linear regression analysis was used to investigate the relationships between body size of nematodes within each trophic group and their heavy metals content.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


The findings obtained from this study provide evidence that the oxidized layer augments the bioactivity of the shape memory alloy. This augmentation was substantiated through the spontaneous biomimetic deposition of apatite from a simulated body fluid (SBF) solution. Furthermore, the modified surface exhibited improved osteoblast proliferation, and enhanced platelet adhesion and activation. This proposed surface modification strategy holds promise as a prospective solution to enhance the biocompatibility and bioactivity of NiTi shape memory alloy intended for prolonged use in bone implant applications.The present study elucidates the impact of glow discharge oxidation within a low-temperature plasma environment on the bioactivity characteristics of an NiTi shape memory alloy. The properties of the produced surface layers, such as structure (TEM observations), surface morphology (SEM observations), chemical and phase composition (EDS and XRD measurements), wettability , and the biological response of osteoblasts and platelets to the oxidized surface compared with the NiTi alloy without a surface layer are presented. The presented surface modification of the NiTi shape memory alloy, achieved through oxidizing in a low-temperature plasma environment, led to the creation of a continuous surface layer composed of nanocrystalline titanium oxide TiO This prominence is due to its distinctive attributes, notably shape memory and superelasticity ,2. Theseallic Ni , which iallic Ni .Therefore, the main problem in the application of NiTi alloy in medicine still remains its high nickel content (over 50% Ni), which although present in trace amounts in the human body, can cause allergies when the safe daily dose approx. 200–300 μg) is exceeded, and exhibit cytotoxic and even carcinogenic effects ,17,18,190–300 μg In pursuit of improving resistance against corrosion and tailoring the biological characteristics of the NiTi alloy to suit specific applications, various surface engineering methods are employed, such as oxidation in air and steam, electrochemical and laser methods, ion implantation, glow discharge treatments, PVD and CVD methods, and deposition of carbon, ceramic or polymer coatings ,25,26,27Our prior investigations have demonstrated that enhancing corrosion resistance, biocompatibility, and diminishing the release of nickel ions into the biological milieu can be accomplished through the application of low-temperature plasma treatment techniques under glow discharge conditions ,25,26,27In light of the above, this work aims to investigate the impact of the glow discharge oxidizing process in low-temperature plasma on the bioactive properties of a shape memory alloy. The assessment is performed via incubation in SBF (simulated body fluid) solution and examination of the resulting apatite layers. A method of producing layers at temperatures up to 300 °C is presented, which guarantees the preservation of the specific properties of NiTi alloy, i.e., the effect of shape memory and superelasticity . In addi2 atmosphere with 99.999% purity).The researched material consisted of an NiTi shape memory alloy with a chemical composition of 50.8 at.% Ni (Ti—rest). Disc-shaped samples with a diameter of ø8 mm and a thickness of 1 mm were used in the tests. The samples were mechanically ground with up to 1200-grit sandpaper and degreased in acetone. The glow discharge oxidizing process was conducted at a temperature of 290 °C for 60 min in oxygen plasma at a chamber operating pressure of 1.6 mbar. The temperature was controlled by a thermocouple. Before oxidizing, the specimens were heated to 290 °C in nitrogen plasma  FEG transmission electron microscope (TEM)  capable of working in scanning mode (STEM). The nanoscale local chemical analysis was performed with an integrated EDAX energy dispersive microanalysis (EDS) system .The surface morphology of the samples before and after incubation in SBF solution was imaged using a scanning electron microscope  with an acceleration voltage of 15 keV in SE (secondary electron) mode. An EDS (Energy-dispersive X-ray spectroscopy) attachment was used for analysis of the chemical composition of the biomimetic apatite layers on the sample surface after incubation.The XRD measurements were performed on a Bruker  D8 Advance diffractometer, equipped with parallel beam optics and a Cu Kα radiation (λ = 0.154056 nm) source that operated at 40 kV and 40 mA at room temperature. The scan optics consisted of a Göbel mirror on the side of the incident beam and twin secondary Soller slits in the diffracted beam. The measurements were performed with a fixed angle of incidence of the primary beam equal to 3°, over the 2θ range, between 20° and 80°, with a step size of 0.025° and a 3 s counting time per step.The wettability test was performed with a goniometer—Contact Angle System OCA 20 . Measurements were made at room temperature for two liquids: distilled water and diiodomethane. After applying a drop of 0.4 μL to the surface of each sample its image was recorded immediately after stabilization. Average values and standard deviations of the contact angles were calculated from 5 measurements performed for each sample. The profiles of the droplets were analyzed using dedicated SCA20 software. Based on the obtained values of contact angles for distilled water and diiodomethane, the surface free energy was calculated for each sample using the standard Owens–Wendt method .2 at 37 °C. The medium was changed every 48 h. Osteoblasts suspended in the incubation medium at a concentration of 3 × 104/500 μL were applied onto NiTi samples in the initial state and after glow discharge oxidizing. The cells were then incubated for 24, 48 h and 6 days at 37 °C. Following incubation, the culture was rinsed with a phosphate buffered saline solution , and the settled cells were fixed in 4% glutaraldehyde for 30 min at 4 °C before rinsing in a cacodylic buffer, in accordance with the procedure for preparing biological material for examinations employing a scanning electron microscope. The prepared specimens were vacuum-coated with a ca. 10–15 nm layer of gold using a JFC-1200 JEOL fine coater. They were then tested under a scanning electron microscope  with an acceleration voltage of 5 keV in LEI (lower secondary electron image) mode.Studies of osteoblast proliferation on the titanium oxide layers produced and the NiTi alloy were conducted with the use of Normal Human Osteoblasts . The cells were grown in an osteoblast basal medium enriched with fetal bovine serum (50 mL/500 mL medium), ascorbic acid (0.5 mL/500 mL medium), GA-1000 (0.5 mL/500 mL medium) antibiotics , in a humid atmosphere of 95% air and 5% COAdhesion and aggregation tests of blood platelets on the surface of NiTi alloy in the initial state and after producing an oxidized layer were performed with the use of platelet rich plasma (PRP), prepared using the blood of healthy donors. The plasma was incubated on the surface of the samples for 2 h at 37 °C. After this time, the non-adhering cells were rinsed off, while the adhering ones were fixed in 4% glutaraldehyde for 30 min at 4 °C, rinsed in a cacodylic buffer and dehydrated and observed under a scanning electron microscope according to the same procedure used for the osteoblast cultures.The number of osteoblasts, as well as platelets and their aggregates, was measured using morphometric CellSens  software. The results are presented as means and standard deviations. Statistical analyses were performed using the Fisher and Student’s t-tests at a significance level of α = 0.05.2 rutile-type with a nanocrystalline structure. The thickness of the titanium oxide layer is approx. 40 nm. The chemical composition of the layer is also confirmed by the linear distribution of elements obtained from EDS (Images from the transmission electron microscope (TEM) show the cross-section of the oxidized surface layer produced at low-temperature plasma on the NiTi shape memory alloy a. As we from EDS b.2 layer was extended to 30 days. Surface images (Scanning electron microscope (SEM) observations show the surface morphology of NiTi samples in the initial state a,a’,c,c’e images a–d as wee images e after 3e images . The rat2 surface layer, a multiphasic composition of calcium phosphates consisting of calcium pyrophosphate dihydrate Ca2P2O7·2H2O (known as CPPD) and hydroxyapatite was formed (8.8(PO4)6(OH)1.92 which is often found with biomimetic compounds formed naturally when samples are soaked in solutions resembling human body fluids and is referred to as bone-like apatite. This would explain the observation that although the EDS analysis shows much more Ca and P after 14 days of incubation on samples with the oxide surface layer than on the non-oxidized samples, the Ca/P ratio is lower, indicating more CDHAP  produced, which is in line with the SEM observations.Usually, the individual components of different types of calcium phosphate compositions are mixed together and strongly integrated with each other, and therefore, the X-ray diffraction technique is very helpful in identifying phases . XRD anas formed . DatasheThe contact angle of NiTi samples increased after low-temperature oxidizing, which means decreased wettability for both polar (distilled water) and non-polar liquids (diiodomethane) . Conside2 oxidized layer compared with the non-oxidized NiTi samples. The number of thrombocytes was much higher for samples with oxidized layers, and furthermore, they formed numerous aggregates, which was revealed both by microscopic observations of adhered cells using a scanning electron microscope (SEM)  a–d and bPlatelet activation was assessed on the basis of their morphology according to the classification described in the literature . The plaOsteoblast proliferation was preserved on both sample types and within the first 48 h they proliferated at a similar rate. Between days 2 and 6, osteoblast growth was inhibited on the samples in the initial state, while on samples with an oxidized layer, they continued to proliferate at only a slightly reduced rate, as evidenced by the slope of the curve in the graph . As a reThe morphology of osteoblasts growing on samples with and without an oxidized surface layer was normal and slightly changed during the experiment . After 2The NiTi shape memory alloy, being self-passivating, is considered a material with good biocompatibility ,39,40. H2 (rutile), which is the most thermodynamically stable polymorphic variety of titanium oxide [2 layer of titanium oxide increases adhesion to the NiTi substrate. Those studies investigated amorphous carbon coating deposition. Our current study shows that the TiO2 layer also ensures faster surface deposition of an apatite layer composed of calcium phosphates.The produced surface layers were characterized by a nanocrystalline structure of titanium oxide TiOum oxide . The maium oxide ,24. Moreum oxide , it was 2 + TiN) layers with a thickness of several dozen nanometers and produced on NiTi alloy at different temperatures. It was found that during glow discharge processes carried out at temperatures above 300 °C, Ni4Ti3 phase precipitates appear in the structure of the alloy [2N sublayer [The use of a glow discharge process temperature below 300 °C made it possible to produce surface layers that retain specific properties of the NiTi alloy, i.e., shape memory and superelasticity, which was previously demonstrated ,59 on thhe alloy . These psublayer , as wellsublayer  x(PO4)6−x (OH)2−x is found, most often also containing other ions . In this respect, biomimetic CDHA is more like an inorganic composition of natural bone, which also shows a lower Ca/P ratio than synthetic bone. Therefore, CDHA, which has a higher specific surface area than HA or tricalcium phosphate (TCP) [Bioactivity, defined as the ability to form a biomimetic bone-like apatite layer in the environment of solutions simulating human body fluids, is especially important in the context of the long-term use implants. There are numerous studies confirming that in vitro studies utilizing the biomimetic method also produce results in vivo, as well as a palpably better connection between the bioactively enhanced material and the bone . This iste (TCP) , exhibitte (TCP) , with pote (TCP) ,66,67,68te (TCP) . They arte (TCP) .2P2O7·2H2O (CPPD) was also found in the apatite layers deposited on the samples, which although less popular, has recently been intensively studied as a material of biological interest [E. coli (74% growth inhibition) and S. aureus (48% growth inhibition) [In addition to the observed deposition of CDHA, calcium pyrophosphate dihydrate Cainterest . The resinterest , as wellibition) . It was ibition) .Better proliferation of such cells as osteoblasts, mesenchymal cells, or platelet adhesion in vitro suggest higher in vivo bioactivity. It is known that platelets contain growth factors, which ensure better healing as well as greater growth of various types of cells. PRP is used as a recovery agent for musculoskeletal injuries. The results presented in this paper are consistent with these observations since the material with the oxidized layer was characterized by both increased proliferation of osteoblasts, and platelet adhesion and aggregation. In our study, osteoblast proliferation was similar for the first 48 h for both types of material. Only after 6 days of proliferation can a statistically significant difference be seen in favor of samples with a surface layer. This is due to the greater biocompatibility of the titanium oxide surface and probably the absence of the cytotoxic effect of nickel. This is consistent with studies from the literature ,20 whichThe response of cells, as well as the bioactivity of the surface, is certainly influenced by many factors, one of them undoubtedly being the wettability of the surface. The findings of this study align with previous literature findings suggesting that surfaces with lower surface energy and increased hydrophobicity promote platelet adhesion ,79,80,81The chemical composition, phase composition, and surface wettability are important, yet are not the sole factors determining material surface interaction with the biological environment. Surface topography and surface charge are additional significant factors that can influence cell adhesion ,90 but w2 (rutile). The low temperature in this process ensures the preservation of the alloy’s specific properties inherent to shape memory. Our research has demonstrated that this newly formed oxide layer significantly heightens the bioactivity of the NiTi shape memory alloy. This was substantiated by the rapid and spontaneous biomimetic deposition of apatite from a simulated body fluid (SBF), a phenomenon that occurred more expeditiously on the modified surface compared with the unmodified material. Additionally, our investigations revealed enhanced osteoblast proliferation on the modified surface, as well as increased adhesion and activation of platelets obtained from platelet-rich plasma (PRP). Furthermore, the presence of hydroxyapatite among the calcium phosphates within the biomimetic layer formed in vitro holds promise for improved in vivo bioactivity. This optimism is derived from the fact that the SBF solution utilized closely mimics both the composition and pH of human body fluids. Consequently, our proposed surface modification presents a prospective avenue for enhancing the biocompatibility of NiTi alloy employed in the development of long-term bone implants.In this study, we have introduced a surface modification approach for the NiTi shape memory alloy. This modification involves oxidation conducted under glow-discharge conditions in a low-temperature plasma environment, yielding a continuous nanometer-thick layer of nanocrystalline titanium oxide TiO


To explore the impact of virtual reality (VR) training on anti-fall ability and bone mineral density (BMD) among elderly patients admitted to a healthcare institution.n = 25) and control group (n = 25). In VR group, the virtual reality rehabilitation training system was used for training, while control group was treated with traditional fall prevention exercise intervention. The changes of Berg Balance Scale (BBS), timed up and go test (TUGT), functional gait assessment (FGA), bone mineral density (BMD) and falls during 12 months of training were compared between the two groups.People (aged 50) with osteoporosis in an elderly care institution in Anhui Province June 2020 to October 2021 were selected and randomly divided into VR group (P < 0.05). However, there was no significant difference in the lumbar spine and femoral neck BMD between the two groups 6 months after the intervention. The femoral neck and lumbar spine BMD of the VR group improved, and it was significantly higher than that of the control group 12 months after the intervention. Nevertheless, there was no significant difference in terms of the incidence of adverse events between the two groups.BBS and FGA were positively correlated with BMD of the lumbar vertebrae and femoral neck, and TUGT was negatively correlated with BMD of the lumbar vertebrae and femoral neck. After 12 months of training, the BBS score, TUGT evaluation and FGA evaluation of the two groups were significantly improved compared with those prior to training (VR training can improve anti-fall ability and increase femoral neck and lumbar spine BMD and can effectively prevent and reduce the risk of injury among elderly people with osteoporosis. With the increasing aging population worldwide, osteoporosis has become a major social issue, and most patients with osteoporosis develop different complications . MoreoveCurrently, pharmacological intervention is the main option for treating osteoporosis among elderly individuals . The VR In elderly healthcare institutions, people without family support are more likely to experience accidental falls, thereby causing fractures. With improvement in science and technology by leaps and bounds, the development of novel technologies for smart elderly care has become a novel trend in response to the global challenges of aging . The curThis research enrolled 50 patients with osteoporosis admitted to an elderly healthcare institution in Anhui Province, China, from June 2020 to October 2021. Moreover, it was designed and conducted by the Department of General Medicine, Bengbu Medical College. All participants provided a written informed consent form, and the study was approved by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College and was performed in accordance with the principles of the Declaration of Helsinki.The inclusion criteria were as follows: (1) individuals aged ≥ 65 years, (2) those who can walk independently or with assistive devices, (3) those with a lower limb muscle strength of ≥ level III and sitting and standing balance of ≥ level II , (4) thon = 25) and VR (n = 25) groups using the random number table method. Among them, 11 presented with high blood pressure, 10 with diabetes, 8 with respiratory disease, 3 with heart disease, and 2 with digestive system disease .In control group, subjects performed routine fall prevention exercises under the leadership of an instructor, including medium-intensity aerobic gymnastics and apparatus exercises, with 50 min each time, 3 times per week, lasting for 12 months. The goal is to achieve core muscle training, lower extremity muscle strength, balance and gait function training.In VR group, a VR rehabilitation training system  was used for training. The subjects wore eye masks and were received 3 kinds of VR sports games: skiing, diving, and running. The training regimen was 3 times per week, 50 min each time , with total training period of 12 months.The International Physical Activity Questionnaire-Short Form (IPAQ-SF) was used to measure Physical activity levels before training .All subjects were tested for anti-fall parameters and BMD before training, 6 months and 12 months post training.Anti-fall parameters include: Berg balance scale (BBS); Timed up and go test (TUGT); Functional gait assessment (FGA).Bone mineral density (BMD) measurement: The BMD of patient’s femoral neck and lumbar vertebrae were measured using a dual-energy X-ray absorptiometry (Hologic Discovery W series). The total spine and Femoral Neck least significant change (LSC) were 2.6% and 4.1%, respectively. And all operators were professionally trained and obtained the training certificate issued by The International Society for Clinical Densitometry.During the training period, all subjects were observed to count the number of falls and the fracture situation after the fall were tracked.P-value of < 0.05 was considered significant.SPSS 22.0 was used for data entry and statistical processing, and GraphPad Prism 8.0 was used for graphing. Percentage changes of data were expressed as mean ± standard deviation (P > 0.05) . The TUG of the VR group decreased significantly compared with that of the control group . The BBS and FGA scores of the VR group increased significantly 12 months after the intervention compared with those of the control group  The TUG score of the VR group decreased significantly compared with that of the control group   . The lumbar spine and femoral neck BMD of the VR group increased significantly 12 months after the intervention compared with that of the control group (4.8%, 95% CI 1.2–8.4, After training, within a follow-up period of 12 months, two subjects in control group had a fall, one of whom experienced a distal radius fracture after falling, while in VR group, one subject experienced a fall but without fracture occurred.The current study aimed to evaluate the effect of exercise applied by VR techniques on improving anti-fall ability and lumbar spine and femoral neck BMD among elderly individuals admitted to a healthcare institution. Results showed that exercise applied by VR techniques was more effective in improving the anti-fall ability and BMD of elderly patients than traditional training. To the best of our knowledge, the number of similar studies was extremely limited. This research innovatively applied exercise applied by VR techniques in the training of elderly individuals admitted to a healthcare institution. Moreover, changes in anti-fall ability and BMD were evaluated, and a novel approach for the treatment of elderly individuals with osteoporosis admitted to a healthcare institution was introduced.Studies have indicated that , 13 an iElderly individuals presented with decreased athletic ability, including coordination, balance, muscle strength, and speed , 17. TheWith aging, bone fragility increases, and BMD decreases. These factors mainly cause fractures. Previous studies have found that adverse factors including decreased exercise capacity and muscle strength affect BMD among elderly individuals , 24. MorVR intervention can cause vertigo symptoms, which can be relieved by removing the headset. This type of vertigo is attributed to the conflict between what your eyes see and the information felt by the vestibular system of the inner ear . ElderlyThe current study had several limitations. That is, the follow-up period was short. Nonetheless, improvement in BBS, TUGT, and FGA scores was consistent with that of previous studies. Hence, there was no significant difference in the incidence of adverse events between the VR and control groups. Nevertheless, the current study also had several strengths. That is, the effect of VR training on BMD among elderly individuals was investigated, and no similar study has been conducted thus far.In conclusion, the use of VR technology in sports training can increase BMD and improve anti-fall ability among elderly patients with osteoporosis admitted to healthcare institutions. Moreover, it can be beneficial for the promotion of smart elderly care during the global health crisis.


Lig-FC is a network depolymerization mechanism for the rapid primary devolatilization of mineral-free lignins that has already been validated with test data on 16 lignin samples. This paper expands the validation with an additional 13 lignins, including cases that applied different lignin preparations to the same feedstock. The validations reported here cover 27 mineral-free lignins for temperatures to 1150 °C, heating rates from 30 to 8000 °C/s, contact times after heatup to 90 s, and pressures from vacuum to 0.13 MPa. Lig-FC accurately depicts the impacts of lignin quality, heating rate, temperature, contact time, and pressure on the major products and oils’ molecular weight distributions (MWDs). All raw lignins contain abundant oil precursors that are released as oils via flash distillation as soon as a flow of noncondensables carries them into the free stream. Consequently, lignin MWD is an essential aspect of lignin constitution because it determines the inventory of inherent volatile chains subject to unhindered flash distillation. Lighter lignin MWDs have larger inherent inventories and therefore produce more oils than heavier MWDs at the onset of devolatilization. Oil yields diminish and char yields increase for progressively heavier MWDs and heavier mean monomer weights and for lignins with relatively less H and more O compared to C. The mechanism’s ultimate application is to accurately describe the release of gaseous fuels and chemical intermediates from the lignin component in any biomass form during suspension firing in pyrolyzers, furnaces, and gasifiers, and in the fluidized suspensions in atmospheric and circulating fluidized bed combustors, and fluidized bed pyrolyzers, and gasifiers. Lig-FC’s formulation is presented in the Supplemental Material to [lig-FC over the full domain of mineral-free lignin characteristics, including any of the major preparation processes, any lignin MWD, and the full range of lignin compositions. However, it does not modify the theory or any of the detailed interpretations of the validation database in [lig-FC appears in [This paper expands the validation of a reaction mechanism for the devolatilization of mineral-free lignins called “erial to  along wiabase in . Consequpears in , and thepears in . Readerspears in  to comprlig-FC were initially formulated as part of a two-component devolatilization mechanism for whole biomass called “bio-FLASHCHAIN®” [lig-FC was recently used to demonstrate an essential role for the flash distillation of oils during the devolatilization at atmospheric pressure of 14 lignins representing a broad range of lignin MWDs and chemical compositions [lig-FC with an expanded submodel for lignin constitution and additional structural features and reaction processes and validate simulation results with a much more extensive database of test results that covers most of the commercial operating domain.The noncatalytic, spontaneous channels in HCHAIN®” . This fiositions . The preThe next section briefly Introduces the vocabulary for the main elements of the theory along with essential aspects of the lignin constitution submodel; much more detail is given in . After tlig-FC, the essential aspects of lignin devolatilization are: (1) reactions that randomly rupture monomers and thereby depolymerize lignin macromolecules over a broad fragment distribution; (2) bimolecular recombination reactions that coalesce the shortest, most volatile macromolecules into longer macromolecules; (3) fragmentation statistics to describe the probability that given monomer ruptures and recombinations produce fragments of a certain size; (4) a cascade of charring reactions that inhibit subsequent chain ruptures while bound monomers decompose sequentially into semichar units, char units, and annealed char plus noncondensable gases; and (5) the flash distillation analogy (FDA) whereby light fragments in the condensed phase vaporize into the escaping noncondensable gases, depending on the molecular weight, temperature, and pressure [2, light oxygenated gases (OXYs), H2O, and H2; the yields and elemental compositions of the solid residue and bio-oil; and the MWD of bio-oil.According to in size;  a cascadLig-FC describes only primary lignin devolatilization and omits all secondary processes. Primary devolatilization exclusively entails chemistry in only the condensed solid phase, whereas secondary chemistry comprises the succeeding chemistry among the gaseous reaction products of primary devolatilization. Lig-FC would normally be coupled with distinct reaction mechanisms for conversion of both volatiles and char beyond the suspended biomass particles in simulations of any biomass utilization technology. In testing, secondary chemistry is avoided by imposing heating rates at least as fast as 5–10 °C/s and by sweeping the primary products away from hot surfaces in an inert gas stream.lig-FC, a rudimentary model of the initial chain distribution is assigned by fitting the proportions of reactant, intermediate, and volatile chains to either the degree of polymerization (DP) or to number-average (Mn) or weight-average (Mw) lignin molecular weights. As developed elsewhere [j is the number of j-mers divided by the initial number of monomers and p(0) is the probability for intact monomers in raw lignin. With this function, a lignin j-mer is depicted as a sequence of j − 1 intact monomers with a broken half-unit on each end. Marathe et al. [w for 14 lignins that were first used to specify p(0), which in turn specified the initial lignin MWD from Equation (1). These lignin MWDs are compared in In lsewhere , the sime et al.  reportedlig-FC are the lignin MWD , monomer compositions from an ultimate analysis, mean monomer weights, and ash contents and compositions . Table e et al. , the comw-values for the 14 tested lignins [The validation database contains six laboratory studies that collectively determine complete product distributions including oil MWDs, including the study featured in the original validation work because it includes M lignins . Marathe lignins  used a ww of 4660 g/mol into three fractions with Mw of 820, 2390, and 6160 g/mol. Tests monitored the partitioning of these fractions into char and a partial gas mixture of CO2, CO, light gaseous hydrocarbons (GHCs), and H2. The differences to close mass balances were reported as “oils” when they actually are sums of oils, H2O, and OXYs. Samples of 100 mg were heated in a quartz U-tube that was inserted into a tube furnace at 400, 500, and 600 °C for 90 s. Reactor dimensions were not reported, so the heating rate cannot be estimated; the simulations are based on 100 °C/s. Moreover, the reported char yields at 400 °C vary from 0 to 78.8 wt.% across the four fractions, and three of four are lower than the char yields for 500 °C; both features are impossible. There are no irregularities in the reported yields for 500 and 600 °C, so only the dataset for 400 °C is ignored. The reported ultimate analyses for the four lignins are within 1.5 daf wt.% for C and O, so the simulations have the same parameter set for these four lignins. Chen et al.  extracte2O, CO, CO2, OXYs, H2, and GHCs across the domain of operating conditions, plus a few elemental compositions for oils and char. Mass balance closures on individual tests are generally within ±5% across the entire database, and in the three tests with oil and char compositions, C/H/O balances were closed to within ±4%. The study’s greatest limitation is that secondary volatiles pyrolysis was unregulated, as there was no sweep stream to remove oils from the reactor hot zone before they could decompose or any other means to quench the volatiles as soon as they escaped from the lignin. The reported product distributions clearly display attributes of secondary pyrolysis, which become more pronounced for progressively hotter temperatures. In the validation work, the threshold temperature for negligible oil decomposition during heatup at 1000 °C/s is estimated as 750 °C, whereas all char yields were qualified. Nunn et al.  used a WThermal histories in these simulations are based on the reported heating rate and reaction temperatures. However, the mesh and sample were not force-quenched; rather, they cooled convectively at approximately −200 °C/s. Preliminary calculations determined that adding a 150 ms IRP for tests that imposed immediate cooling after heatup gave comparable incremental yields after the end of the heating period. In all simulations of this dataset, this 150 ms IRP was added to the reported IRPs in the thermal histories.Zhou et al.  used a W4 °C/s to 350–750 °C with 20 s IRP. It is not clear if volatiles were swept out of the hot zone throughout the test although there are no clear indications that oils decomposed at the hottest test temperatures. Tests monitored the partitioning of the lignins into char and a partial gas mixture of CO2, CO, and GHCs. The differences to close mass balances were denoted as “oils plus H2O” when they actually are sums of oils, H2O, H2, and OXYs. The most distinctive feature of this dataset is that the suite of lignins represented three lignin preparations applied to the same hardwood and softwood samples. Both woods were first extracted in a toluene/ethanol mixture, then the lignins were collected with treatments of dioxane/HCl, Klason hydrolysis, and organosolv in ethanol/H2SO4. Since the reported ultimate analyses for each lignin pair from the same recovery process were substantially different, distinctive parameter sets were assigned for each sample to fit the reported partitioning of lignins into char and aggregate oils. No lignin MWD information was reported, so the Mw-values were estimated as 2000 and 2500 g/mol for softwood and hardwood lignins, respectively, except that the Klason hardwood lignin has an Mw of 3700 g/mol.Custodis et al.  used a pThese six datasets are complementary: Marathe et al. thoroughly characterized oil MWDs from 14 lignins and Nunn et al. thoroughly characterized all the major product yields and compositions, albeit for a single lignin. Chen et al. expanded the range of lignin molecular weight. The tests from Pecha et al. and Zhou et al. substantially extend the range of heating rates in the validation database. Custodis et al. characterized the impacts of different lignin recovery processing on hardwood and softwood. All samples in this database have less than 0.1 wt.% ash and are regarded as mineral-free. Two other datasets qualified for lignin primary devolatilization are interpreted elsewhere with the same parameter ranges used here [The kinetic parameters assigned for monomer rupture, recombination, monomer decomposition, semichar decomposition, and annealing are reported in , as are 2, CO, GHCs, and H2, and a mixture of oils, H2O, and OXYs assigned to close the mass balances. The simulated products are grouped in the same ways, except that the right panel also shows just the simulated oil yields for comparison. Since the reported ultimate analyses for the whole lignin and the three fractions are nearly the same, the simulations used a single parameter set for the four samples. At both temperatures, char yields increase and condensable yields decrease for progressively heavier lignins while the partial gas yields are hardly perturbed. Char yields diminish and partial gas yields increase by the same amounts for the hotter temperature, which reflects char decomposition and annealing. Normally, 600 °C is too cool for annealing, but due to the extended IRPs in these tests, it does make an appreciable contribution at the hottest temperature. The simulated condensable yields for both temperatures are indistinguishable because oil production finishes by the time the lignins reached 500 °C for this heating rate of 100 °C/s. Similarly, the measured condensable yields are the same within the measurement uncertainties. The simulated oil yields are somewhat less sensitive to lignin molecular weight than the condensable yields but only for weights heavier than 4500 g/mol. This transition occurs at the weight that has relatively few inherent oil precursors in the raw lignin, so that monomers must rupture to depolymerize the intermediate chains into the volatile size range before oils can be produced. Since parameters were adjusted to match reported yields for every distinctive lignin composition in the validation database, the model results will be called “simulation results” rather than “predictions”. W-values for all samples appear across the top of the figure. Note that the MW-values are for discrete sample numbers and do not determine a continuous variation in this parameter along an axis. Each of the four triplets are arranged as whole, light, and heavy samples from left to right. Yields were not reported for sample 9 under vacuum and for samples 6 and 7 at 0.1 MPa. The prominent impact of lignin MWD is apparent in two ways in lig-FC is especially significant because it is evident in the measured char yields for all four triplets, despite large variations in composition among these four samples  and is neither statistically significant nor unique, because a single dataset cannot determine so many parameters with stringency. The simulated dynamics of gas release are consistently faster than in the reported gas release histories and shift toward cooler temperatures by about 50 °C. This flaw is inconsequential because it was also seen in the cellulose devolatilization histories from this older pyrolysis system [The complete distribution of products reported by Nunn et al.  is the bboratory . The sims system  compared2 yield is 1.2 wt.% at 750 °C, whereas H2 was not measured. It is intended to represent both molecular H2 and the hydrogen in the reported ultimate yields of 0.8 wt.% CH4 and 0.5% C2 GHCs, because GHCs are omitted from the noncondensables in lig-FC. It exceeds the hydrogen in the reported GHCs by slightly less than 1 wt.%. The simulated H2O than the measured values at this temperature  oil yields diminish for progressively heavier lignin MWDs while char yields increase; (ii) oil MWDs shift toward heavier weights for progressively heavier lignin MWDs; (iii) oil yields are markedly greater under vacuum than at atmospheric pressure; (iv) oil MWDs become heavier for progressively lower pressures and hotter temperatures; and (v) oil yields increase for progressively hotter temperatures, especially under vacuum. Since lignins in each triplet in Marathe et al.’s dataset and the four lignins tested by Chen et al. have the same compositions, only seven parameter sets accurately interpreted the partitioning of 18 distinctive lignins, and only the frequency factors for recombination and for the first two steps in the charring cascade were varied. These validations are especially stringent because they were supported by measured Mw-values for all 18 lignins and collectively cover a broad domain of operating conditions. Only the impact of the lignin recovery process remains inconclusive. Lig-FC also accurately depicts complete distributions of the major noncondensables and reasonable elemental compositions of oils and char, although many more complete product distributions for diverse lignins need to be reported to finalize the stoichiometric coefficients.4 °C/s, while temperatures for the maximum production rates increase from 340 to 670 °C. The temperature shifts are responsible for the substantially heavier oils for progressively faster heating rates, indicated in n-values for the ultimate cumulative oil sample, which surge for heating rates faster than 300 °C/s. Over this range of heating rate, ultimate oil yield increase from 26 to 62 wt.%, while char yields diminish from 37 to 15%. The gas yields fall in tandem with the char yields. Unfortunately, measured lignin partitioning across such a broad range of heating rate, with all else the same, has not yet been reported, although one evaluation reported elsewhere [The only variable operating condition not represented clearly in the validation database is heating rate, which was varied over a wide range but for different lignins, pressures, and contact times. The simulation results in Lig-FC depicts the distinctive devolatilization behavior of individual lignin samples in terms of the lignin MWD, the elemental compositions of monomers, the average monomer molecular weight, and mineral levels. Mineral catalysis was excluded from this study and characterized separately [w-values lighter than 2500 g/mol that have half or more of their macromolecules in the volatile size range [w is 1000 g/mol and the other has 3600 g/mol . With the light sample, the volatile chain inventory abruptly falls to 25 wt.% in only 150 ms. Such a small inventory suppresses bimolecular recombination, so that the intermediate inventory surges at 100 ms to only 10 wt.%, then gradually reaches 15% when all volatile chains have been converted. With the heavy sample, the volatile inventory first increases to 45 wt.% as intermediates are ruptured, then gradually diminishes through 600 ms. Since volatile chains persist at elevated levels, recombinations counteract the rupture of intermediates at 200 ms and then sustain the approach to the saturation level of 25 wt.% for the ultimate char yield. parately , whereasparately . The keyze range . With sos sample ). Since These differences in the MWDs of the condensed phase strongly affect oil production. With the light sample, oils are produced as soon as gases are released, first via monomer rupture and decomposition and then via recombination. From the onset of gas production at 75 ms, flash distillation releases oils from the near-complete inventory of volatile chains. By the time recombination begins at 100 ms, nearly half the ultimate oil yield has already been released. Thereafter, recombinations and the charring cascade compete more effectively with flash distillation to retard oil production and to grow the inventory of intermediate chains that comprise the ultimate char. The transition from unhindered flash distillation of inherent volatile chains to a competition among flash distillation, recombination, and the charring cascade is also evident with the heavier sample. However, unhindered flash distillation from 75 to 100 ms produces less than 10 wt.% oils vs. 30% with the light lignin, because the inherent volatile chain inventory is only one-third of the lignin vs. almost 100% with the light lignin. Thereafter, the competition among flash distillation, recombination, and charring drives oil production to the ultimate saturation level. However, it does not compensate for the different outcome of unhindered flash distillation, so the ultimate oil yield is only 56 wt.% vs. 70 wt.% for the light lignin. Lignin MWD is an essential aspect of lignin constitution because it determines the inventory of inherent volatile chains subject to unhindered flash distillation. Lighter lignin MWDs have larger inherent inventories and therefore produce more oils than heavier MWDs at the onset of devolatilization. These differences largely vanish during the succeeding competition among flash distillation, recombination, and the charring cascade.n shifted from 480 to 510 g/mol and Mw shifted from 650 to 690 g/mol. The mean monomer weight is an essential aspect of lignin constitution because it directly factors into the instantaneous weight of the vaporizing volatile chains throughout devolatilization. Variations in lignin elemental composition were assessed elsewhere [The impact of variations in the monomer weight was assessed elsewhere  in simulFinally, one of the most striking features in the validation database is the impact of pressure on the reported oil yields in (1)Lig-FC identifies the essential reaction mechanisms in primary lignin devolatilization as depolymerization via monomer rupture in competition with charring via a spontaneous three-stage cascade and bimolecular recombination and flash distillation of oil precursors from the condensed phase.(2)lig-FC, the first gases form by monomer decomposition and rupture. Flash distillation moves inherent oil precursors from the condensed phase into the stream of noncondensables, which carries them away as product oils. Hence, the induction of primary lignin devolatilization does not entail depolymerization; only monomer decomposition and flash distillation of the inherent oil precursors are required.In (3)W up to 2500 g/mol have at least half their monomers in inherent oil precursors.Lignin MWD determines the inventory of inherent volatile chains subject to unhindered flash distillation. Lighter lignin MWDs have larger inherent inventories and therefore produce more oils than heavier MWDs at the onset of devolatilization. Raw lignins with M(4)The distinctive devolatilization behavior of individual lignins can be depicted from the lignin MWD, the elemental compositions of monomers, and the average monomer weight. Oil yields diminish and char yields increase for progressively heavier MWDs and mean monomer weights and for lignins with relatively little H and abundant O compared to C.(5)Bimolecular recombination is required to interpret the disparate ultimate oil yields under vacuum and at 0.1 MPa. Suppressed volatility at atmospheric pressure in conjunction with faster recombination reduce the ultimate oil yields to only half of those under vacuum.The expanded validation cases in this study further buttress the conclusions in , which a


On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3′MBR and 5′mcr. The aim of this study was to analyze the correlation of t breakpoint site with clinical characteristics in follicular lymphoma.We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3′MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared.Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3′MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms.Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation. In Europe and USA, it is the second most common type of lymphoma. Follicular lymphoma is considered an incurable disease. It is characterized by an indolent clinical course though it may transform into a more malignant diffuse large B-cell lymphoma.BCL2) gene next to the transcriptional enhancer of the immunoglobulin heavy chain gene (IGH), resulting in BCL2 protein overexpression.7 BCL2 protein is a member of the BCL2 family which consists of pro- and antiapoptotic proteins as well as of proteins not linked to apoptosis. It is localized in the outer mitochondrial membrane and exerts its antiapoptotic function by binding proapoptotic BCL2 family proteins such as BAX and BAC to prevent the release of cytochrome c from mitochondria in the intrinsic apoptosis pathway.8 The translocation is an early event in lymphomagenesis and although on its own likely insufficient, it plays an important role in follicular lymphoma pathogenesis. It results in extended survival of the tumor cells which may cause the accumulation of additional oncogenic genetic aberrations. Follicular lymphoma bears many chromosomal aberrations that vary in number, mostly of unknown or questionable contribution to pathogenesis.10The translocation places the antiapoptotic B-cell lymphoma (11 A commonly used method for translocation detection is Flourescence In Situ Hybridisation (FISH). FISH probes bind to the entire IgH and BCL2 genes thereby indiscriminately detecting translocations at various sites across the BCL2 gene. It has close to 100% sensitivity in the t detection.12 Unlike with FISH, with PCR it is possible to detect the exact breakpoint site, making it indispensable for a study of clinical implications of different breakpoints. PCR is also less expensive and time consuming. However, it does have lower sensitivity of 60–70% as PCR primers identify only short DNA sections.13 Alternatively, multiple primers may be used to amplify and detect different breakpoints. This method has a higher sensitivity of up to 88%.15The t translocation was first detected by karyotypic analysis, which is at present not used for this purpose.H gene segments, whereas on chromosome 18 different breakpoints occur relatively often. Since the breakpoint is usually located outside of the protein coding part of the BCL2 gene, variations in the breakpoint region do not affect the BCL2 protein. In 50% to 65% of cases the breakpoint occurs at the major breakpoint region (MBR) located at the 3′-untranslated region of the BCL2 exon 3. In about 10–20% of cases the breakpoint occurs at the minor cluster region (mcr) located 20 kilobases (kb) from 3′ of the MBR. Additionally, the breakpoint may also be located between the MBR and the mcr, at 3′MBR and 5′mcr subclusters, commonly called the intermediate cluster region (icr).16 The 3′MBR subcluster is positioned 4 kb downstream of the MBR, while the 5′mcr subcluster is positioned 10 kb upstream of the mcr  translocation, the breakpoint location on chromosome 14 is almost invariable in one of the six J the mcr .16The aim of this study was to analyze the correlation of t breakpoint site with clinical characteristics in follicular lymphoma.In this clinical retrospective study, we included 84 patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment between 2013 and 2020 at the Institute of Oncology, Ljubljana and had t detected by PCR prior to systemic treatment. PCR was performed on bone marrow samples as a part of the diagnostic procedure. All patients included in the study signed an informed consent allowing treatment and use of their clinical information and biological material for scientific purposes. The study was approved by the Committee for Medical Ethics of Institute of Oncology Ljubljana (ERIDNPVO-0064/2022).Data regarding treatment protocol and patients’ clinical information were collected from the clinical information system. The following characteristics observed at the time of diagnosis were gathered: gender, age, Ann Arbor stage, grade, presence of B symptoms, FLIPI score, presence of bulky disease , and breakpoint region of the t translocation. Progression-free survival (PFS) was defined as time from the end of the systemic treatment until relapse or end of observation, overall survival (OS) as time from diagnosis until death or end of observation and lymphoma specific OS as time from diagnosis until lymphoma-related death or end of observation. The data were collected on December 20, 2022.InVivo Scribe Technologies, San Diego, CA, USA). This assay amplifies genomic DNA between primers targeting the BCL2 gene and conserved joining regions of the IGH gene. Master mixes for MBR, 3′MBR and mcr detection each contained primers targeting the J region of the IGH gene (JH) and those targeting MBR, 3′MBR and mcr, respectively.DNA was isolated from bone marrow specimens using the QIAamp DNA Blood mini kit . The concentration and the purity of DNA were determined using the Nanodrop spectrophotometer . PCR was performed using IdentiClone™ BCL2/JH Translocation Assay  and consensus JH primer; the mcr master mix contained three mcr primers  and consensus JH primer. Primers design and validation has been described by JJM van Dongen with colleagues.16 Primer sequences with National Center for Biotechnology Information (NCBI) accession numbers are shown in The MBR master mix contained two MBR primers (MBR1 and MBR2) and consensus JPCR products were detected by polyacrylamide gel electrophoresis  and visualized by UV illumination of gels stained with ethidium bromide (0.5 μg/ml). Tested samples were determined as positive for the presence of the t translocation if one or two of the amplified products (bands) within 100–2500 bp range were present. The quality of the input DNA was tested with Specimen Control Size Ladder Master Mix which targets multiple house-keeping genes and generates a series of amplicons approximately 100, 200, 300, 400, and 600 bp long to ensure control of the quality and quantity of the input DNA.Clinical characteristics were compared among the groups defined by the breakpoint region using 1way Analysis of Variance (ANOVA) test and Independent-Samples T-test for numerical and Fisher's exact test for nominal variables. To compare OS and PFS between the groups Log Rank (Mantel-Cox) analysis was performed. p < 0.05 was defined as statistically significant.Among 84 included patients, the group with MBR breakpoint was the most numerous with 63 patients, followed by mcr with 17 and 3′MBR with 4. Female predominance was present in all breakpoint-site groups. Overall, the median age was 61 years, with the mcr group being the oldest. Half of the 3′MBR group and up to one quarter of the 2 larger groups had grade 3 follicular lymphoma. FLIPI score was predominantly 2 or 3 and was lowest in the MBR group. B-symptoms were present in approximately half of the patients in the 3′MBR and mcr group whereas they were less common in the MBR group. Disease stage was highest in the mcr group although stage 4 was predominant in all 3 groups. Bulky disease was mostly absent in all groups with the mcr group having the lowest proportion ,3.Comparing clinical characteristics at diagnosis, a statistically significant difference in stage was found between the MBR and mcr groups p = 0.023). No other significant correlation was established comparing the MBR, mcr and 3′MBR groups or the 2 larger groups only ,3.. No otheAll patients were treated with RCHOP  or RCHOP-like chemoimmunotherapy, followed by irradiation in case of residual disease. Treatment response was defined as complete remission, partial remission, stable or progressive disease, based on the positron emission tomography-computerized tomography (PetCT) 3–5 weeks after the end of systemic treatment. In case of irradiation of residual disease, additional computerized tomography (CT) was performed 3 months after irradiation and was included in final response evaluation. After systemic treatment, patients received maintenance rituximab for 2 years and were subject to a regular follow-up.During observation, 23 patients in the MBR and 9 patients in the mcr group relapsed and none in the 3′MBR group. The Log Rank PFS comparison found no significant difference in PFS between the 3 groups (p = 0.157) or between the 2 larger groups (p = 0.235). Though statistically insignificant, PFS was longer in the MBR group .In the MBR group, 11 patients died, whereas in the mcr group the number of deceased was 5 and no patients died in the 3′MBR group. No significant difference in OS between the 3 breakpoint-site groups (p = 0.426) or the MBR and mcr group (p = 0.351) was observed . LymphomBCL2 gene, nor does it affect BCL2 expression level.17 Nevertheless, a difference in stage between the 2 common breakpoint sites mcr and MBR transpired in our routine clinical data at diagnosis, prompting this study.It is supposed that translocation site in t  translocation bears no prognostic or predictive value as it does not alter the protein-coding part of the antiapoptotic Among 84 included patients, we found MBR breakpoint to be by far the most common with 63 patients, followed by mcr with 17 patients. Only 4 patients had the 3′MBR breakpoint site, making a characterisation of this group difficult.et al. studied clinical characteristics of 236 follicular lymphoma patients with the t translocation, determining five different breakpoint regions, including MBR and mcr. MBR breakpoint was found in 118 and mcr in 11 patients.18 In another study, López-Guillermo et al. determined the BCL2 breakpoint site in 247 patients with indolent follicular lymphoma. They determined breakpoints at the MBR and mcr regions only. MBR breakpoint was found in 175 cases and mcr in 27.19 Compared to the two studies, our mcr group was proportionally the largest with mcr/MBR ratio at 0.27, compared to 0.09 in Weinberg's and 0.15 in Guillermo's study.We only found a few studies treating the subject of this article. In one of them, Weinberg Comparing the groups with different breakpoint region, PFS, OS and lymphoma specific OS were found to be higher in patients with MBR breakpoint site compared to mcr, though the results did not reach statistical significance. Apart from a higher proportion of bulky disease, the MBR group was indeed characterized by a more favorable disease presentation, namely lower grade, smaller proportion of patients with B-symptoms, lower FLIPI score and younger age at diagnosis. Remarkably, the MBR group also had a significantly lower clinical stage compared to the mcr (p = 0.023).18 López-Guillermo compared the MBR and mcr group only and found no significant difference in stage, age, gender, and B symptoms. In contrast to our finding however, he observed a significantly longer PFS in the mcr compared to the MBR group. There was only 1 relapse among 27 patients with mcr breakpoint and 42 among 175 patients with MBR breakpoint. The study of López-Guillermo was indeed performed in the setting of the low-grade follicular lymphoma, with only 3% of patients having follicular lymphoma grade 3 compared to our 22%.19 To obtain more relevant results for this comparison, we conducted the same comparison on our grade 1 and 2 follicular lymphoma, only to find similar results. Taken together, no clear conclusions can be drawn as to correlation between PFS and the t breakpoint region.In the studies of Weinberg and López-Guillermo, no similar findings seemed to transpire. Weinberg compared MBR and “minor breakpoints” group where along with mcr, other breakpoints were included. No significant difference was found in stage, nor in age, B symptoms, FLIPI score. Furthermore, no significant difference was observed comparing PFS and OS between the two groups.In conclusion, we found follicular lymphoma patients with MBR breakpoint to exhibit a more favorable clinical presentation including a higher PFS and OS. Due to our limited sample size and some incongruity in the literature, a larger study would be required to confirm our observation.


Results showed that the mean concentrations of heavy metals in mg/kg were Fe (11.60–34.45), Cu (0.50–8.10), Ni (0.50–1.60), Mn (0.70–25.40), Zn (4.40–15.90), Pb (0.53–10.20), Cd (0.51–0.85), Cr (0.50–1.53) and Co (0.50–1.51). The highest concentrations were found in flour milled using a traditional metallic mortar while the lowest levels were in those samples milled using a wooden mortar. The Fe, Pb and Cd contents of flours produced using the metallic mortar and some commercial mills was found to be higher than the permissible limits set by WHO/FAO. Human health risk assessment showed that there are potential carcinogenic health risks from adults’ intake of heavy metals in maize flour milled using a metallic mortar. Therefore, processing of maize flour needs to be monitored by the relevant statutory bodies in Uganda to minimize the possibility of heavy metal contamination of food products and animal feeds.Consumption of maize and maize-based products contributes a significant percentage to the total food energy intake in Uganda. However, the production of maize-derived foodstuffs is performed traditionally or by small- and medium-scale processors using different processing techniques. This can lead to differences in the quality of these products from processors, raising food safety concerns. In this study, the effects of mechanical processing (milling) methods on deposition of heavy metals into milled maize flour and the associated consumption health risks were assessed. Atomic absorption spectrophotometry was used to quantitatively establish the concentration of iron (Fe), manganese (Mn), zinc (Zn), cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), cobalt (Co) and nickel (Ni) in 100 samples of maize milled using a wooden mortar ( Food is nutritionally considered to be the primary source of essential human micronutrients such as copper (Cu), cobalt (Co), nickel (Ni), iron (Fe), molybdenum (Mo), manganese (Mn), selenium (Se) and zinc (Zn) ,2. HowevFood safety hazards are of biological, chemical and physical nature and may be present at the same time in a given food matrix . They maDietary exposure to HeMs has been extensively studied. However, most studies do not pay attention to potential introduction of these contaminants during food processing. A couple of empirical studies give credit that milling of cereals  expose consumers to leached HeMs ,19,20,21The study was limited to the quantification of HeMs  in maize flour from Mbarara City, Western Uganda . The HeMAll reagents and chemicals used in this study were of high analytical purity  or Sigma Aldrich , and were therefore used in the analytical work without prior purification. Unless otherwise indicated, all solutions were prepared using double distilled water.Dry maize grains (50 kg) were purchased from rural farmers in Kamwenge Sub County, Kamwenge District, Western Uganda. This is an agricultural area with a limited number of automobiles and no heavy industries that can pollute the environment via aerial deposition. In this area, farmers depend on the natural fertility of soils for growing their crops; thus, there is limited pollution through the use of synthetic fertilizers. Maize grains from this area are bought by the business community from Mbarara city who later transport, mill and sell the maize flour within the city.Measured grains (1 kg) were washed and rinsed with distilled water, spread on a plastic sheet to dry indoors to reduce possible aerial pollution (deposition of HeMs). The clean grains (0.5 kg) were pounded into flour using a wooden mortar and pestle without any metallic contact. Similarly, another sample of the washed grains (0.5 kg) was pounded into flour using a metallic mortar and pestle. The two samples generated were used as controls. The remaining grains were milled into flour using selected grain milling machines in Mbarara city, Western Uganda.The maize flour samples for laboratory analysis were collected between the months of December 2022 and March 2023. These were from four different diesel engine and electric powered hammer-type mills. The diesel mills were labeled A, B, C and D while the electric mills were coded E, F, G and H. The location of the mills was as follows: A and E in the Kamukuzi division, B and F in the Kakoba division, C and G in the Nyamitanga division and H and D in the Makenke division.Three samples were collected in polythene bags from each of the mills  at two different intervals during each milling phase, that is, within the first 5 min when the flour first exits on starting and the second interval of sampling was when the flour would be exiting before milling is stopped. A total of 48 samples was collected from diesel engine-powered mills. Similarly, 48 flour samples were collected from the electric motor-powered mills. Two flour samples each were collected for maize processed using wooden and metallic mortars and pestles. Therefore, a total of one hundred (100) maize flour samples was collected and kept in airtight plastic buckets at room temperature in the Chemistry laboratory at the Department of Chemistry, Mbarara University of Science and Technology, Mbarara, Uganda.v/v) in a fumehood for 30 min. They were transferred onto a hot plate for hot digestion which went on until brown fumes were given off from the samples. Then the contents were washed down using distilled water and left to boil to below 50 mL. The digests were cooled and filtered through Whatmann filter papers. The filtrates were transferred into 100 mL volumetric flasks and made up to the mark with deionized water.Analytical flour samples were obtained from pooled maize flour samples using the cone and quarter method. Each analytical sample (3 g) from the cone and quarter method was put on a porcelain crucible and dried in the oven for 24 h at 85 °C. The samples from the oven were removed and cooled in a desiccator to avoid reabsorption of moisture for dry basis analysis. Measured 2.0 ± 0.1 g of the samples were transferred into 250 mL beakers and cold digested with 100 mL of perchloric acid/nitric acid mixture  at 248.3, 279.5, 213.9, 324.7, 232.0, 228.8, 248.3, 238.3 and 357.9 nm for Fe, Mn, Zn, Cd, Pb, Cr, Cu, Co and Ni, respectively ,35.2 > 0.996). The assurance of the quality of the analytical results was addressed through analysis of procedural blanks and spiked samples, whose recoveries were found to be acceptable (range: 98% to 100%). The relative standard deviations of the experiments  varied from 3.5 to 5.0%.The elemental concentrations were established from calibration curves constructed from diluted working standards of 1000 ppm stock solutions of nitrate and chloride salts of the HeMs. The linearity of the curves fell within acceptable limits (RADD), hazard quotient, hazard index and cancer risk for both adults and children [ADD (mg/kg/day) was estimated to discern human exposure through direct ingestion of maize flour as bread or porridge (Equation (1)).C is the concentration of the heavy metal (mg/kg), IR is the ingestion rate = 100 mg/kg/day and 200 mg/kg/day for children and adults, respectively, Ef is the exposure frequency = 365 days/year, Ed is the exposure duration = 58.65 years for an adult Ugandan [ab is the average body weight; as per the clinical guidelines from the Ugandan government, children are those who are 6 years of age weighing 15 kg, while adults were defined as those who are 30 years or older weighing 70 kg [Tae is average exposure time for non-carcinogens = Ef × Ed.The carcinogenic and non-cancer health risk assessments involved the computation of average daily dose  was calculated to establish non-cancer risks from the HeMs (Equation (2)). Since the effects of HeMs can be augmentative, the hazard index (HI) or total HQ was consequently calculated (Equation (3)).e US EPA ,40,41,42−4 indicate unacceptable risks.The carcinogenic health risk (CR) was computed as the incremental lifetime cancer risk for the carcinogenic elements  (Equation (4)). The total cancer risk (TCR) was estimated as the sum of all the cancer risks due to the ingestion of the four heavy metals in maize flour (Equation (5)).ectively . The CR p < 0.05 using GraphPad Prism for Windows .Quantitative data from experimental analyses performed in triplicate were captured in Microsoft Excel  where they were averaged and expressed as means ± standard deviations of replicates. The concentration of the HeMs was compared with the Ugandan standard for maize flour US EAS 44: 2019) [: 2019 4 and thosp < 0.05). The results indicate that the contribution of the metallic mortar was highest with regard to the HeMs Fe, Mn, Zn, Pb and Cu as compared to the other metals (p < 0.05) when compared to the other elements determined. Nevertheless, the concentration of Cd, Pb, Cr, Co and Ni were found to be higher than the WHO/FAO food standards [The effect of different mechanical grinding methods on the deposition and concentrations of some selected HeMs in milled maize from Mbarara city, Uganda was investigated. The concentrations of HeMs in maize flour samples were higher in those ground using the metallic mortar than the wooden mortar . Samples milled using electric motor−powered mills had higher HeMs than those produced using diesel engine−powered mills. However, these differences were not statistically significant (p < 0.05). In comparison to the Ugandan standard (US EAS 44:2019) [On the other hand, maize flour obtained using hammer mills had higher levels of HeMs than that in samples from the wooden mortar  to 1890 × 10−6 for Cd in maize milled with the metallic mortar. For adults, the hazard quotients span from 0.95 × 10−6 (for Cu from maize flour milled by use of the wooden mortar) to 8325.71 × 10−6 for Pb in maize milled with the metallic mortar. Accordingly, the hazard quotients from Pb and Cd, as well as the total hazard quotient, were less than 1, showing that there are no potential health risks from the ingestion of milled maize flour  in the picture, e.g., “10000” should be “10,000”.otal cancer risk values were within the acceptable range of 1 × 10−6 to 1 × 10−4, implying that there are no potential carcinogenic health risks for children who consume the maize flour. For adults, cancer risk values ranged from 0.715 × 10−6 for Cr to 247.690 × 10−6 for Pb. In adults, the individual cancer risk value for Pb in flour milled using a metallic mortar was greater than the safe limit of 1 × 10−6 to 1 × 10−4.For the incremental life cancer risks, the risk values of the individual groups were computed . For theNutritional food quality plays an important role in maintaining human health. However, food and drinking water, along with occupational exposure, are the main routes of exposure to potentially toxic elements in humans. In this study, the concentrations of HeMs in milled maize flours were higher in samples obtained using the metallic mortar than the wooden mortar. However, relatively higher levels of Fe and Zn leached from the wooden mortar into the flour probably because the wooden mortar and pestle are made of natural solid materials which contain phytoavailable levels of geogenic HeMs. The highest concentrations of HeMs were recorded in flour samples obtained using the metallic mortar, though Cd, Cr, Co and Ni occurred as traces. The high levels may be attributed to the presence of these metals as impurities which are added or not removed during metal refining processes. In Uganda, metallic mortars are shared within the neighborhood for preparing small quantities of food and medicinal herbs which cannot be commercially milled. These metallic mortars are possible sources of food contaminants because they are fabricated from rusty and corroded metallic pipes that have been discarded from the Kilembe mines (Western Uganda) and later collected by scrap dealers for recycling. Without any tests for hardness and other chemical properties, local artisans fabricate these pipes into food equipment for use in small scale food industries.For the maize flour obtained using hammer mills, the higher levels of HeMs than that found in samples from the wooden mortar could be because hammer mills are fabricated using mild steel which contains Fe, Mn, Cr and Cu . DependiIn the human health risk assessment, the results of our study agreed with a previous study done in Kampala (Uganda)  in which−4 threshold provided by the US EPA. Another study in Ghana [For the cancer risk assessment, the individual and total cancer risk values for Pb in flour milled using the metallic mortar surpassed the safe limit suggested by US EPA, suggesting potential cancer risks in the exposed population. A similar study  of maizein Ghana  concludeThis study revealed that the milling method used for processing maize into flour had an effect on (enriched) the heavy metal content of the flours. The metals were plausibly deposited into the flour due to frictional wear of the mortars/mill plates during milling. The concentrations of the HeMs in maize flour produced by metallic mortar and commercial mills were found to be higher than the dietary tolerance limits set by the Uganda National Bureau of Standards (for Pb) and the WHO/FAO . Human health risk assessment showed that there are potential carcinogenic health risks from adults’ intake of HeMs in maize flour milled using a metallic mortar. Thus, the processing of maize flour needs to be monitored by the relevant government bodies such as UNBS which are mandated to control quality manufacturing (fabrication) of food processing equipment including mill plates to minimize the possibilities of heavy metal contamination of human food and animal feed.