Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.

Combined medial patellofemoral ligament and medial patellotibial ligament reconstruction in recurrent patellar instability: A systematic review and meta-analysis.

BACKGROUND: The medial patellofemoral ligament (MPFL), along with the medial patellotibial ligament (MPTL) and medial patellomeniscal ligament, aid in the stabilization of the patellofemoral joint. Although the MPFL is the primary stabilizer and the MPTL is a secondary limiter, this ligament is critical in maintaining joint stability. There have been few studies on the combined MPFL and MPTL reconstruction and its benefits. AIM: To look into the outcomes of combined MPFL and MPTL reconstruction in frequent patellar instability. METHODS: By May 8, 2022, four electronic databases were searched: Medline (PubMed), Scopus, Web of Science, and Google Scholar. General keywords such as "patellar instability," "patellar dislocation," "MPFL," "medial patellofemoral ligament," "MPTL," and "medial patellotibial ligament" were co-searched to increase the sensitivity of the search. RESULTS: The pooled effects of combined MPFL and MPTL reconstruction for Kujala score (12-mo follow-up) and Kujala score (24-mo follow-up) were positive and incremental, according to the findings of this meta-analysis. The mean difference between the Cincinnati scores was also positive, but not statistically significant. The combination of the two surgeries reduces pain. According to cumulative meta-analysis, the trend of pain reduction in various studies is declining over time. CONCLUSION: The combined MPFL and MPTL reconstruction has good clinical results in knee function and, in addition to providing good control to maintain patellofemoral joint balance, the patient's pain level decreases over time, making it a valid surgical method for patella stabilization.

Differentiation of Human Wharton Jelly Mesenchymal Stem Cells into Germ-Like Cells; emphasis on evaluation of Germ-long non-coding RNAs.

BACKGROUND: The proliferation and differentiation of stem cells into Germ-Like Cells (GLCs) is mediated by several growth factors and specific genes, of which some are related to long non-coding RNAs (lncRNAs). We have developed a modified differentiation process and identified a panel of GermlncRNAs related to GLCs. METHODS: Human Wharton Jelly Mesenchymal Stem Cells were treated with 25 ng/ml Bone Morphogenetic Protein (BMP)-4 and 10- 5 M all-trans retinoic acid to differentiate them into germ-like cells. To confirm the differentiation, changes in the expression of Oct-4, C-kit, Stella, and Vasa genes were assessed using quantitative Real-Time PCR (qPCR) and immunocytochemistry. QPCR was also used before and after differentiation to evaluate the changes in a lncRNA panel, using a 96-well array. Statistical analysis of the data was performed by SPSS 21. RESULTS: After 21 days of induction, the HWJ-MSCs derived germ-like cells were formed. Also, qPCR and immunocytochemistry showed that the pluripotent Oct4 marker was expressed in the undifferentiated HWJ-MSCs, but its expression gradually decreased in the differentiated cells. C-kit was expressed on days 7, 14, and 21 of differentiation. Both GLC markers of Stella and Vasa genes/proteins were present only in differentiated cells. Of the 44 lncRNA genes array, 36 of them showed an increase and eight genes showed a decrease. CONCLUSION: Our study showed that BMP4 and RA are effective in inducing HWJ-MSCs differentiation into GLCs. In addition, our study for the first time showed changes in the lncRNAs expression during the differentiation of HWJ-MSCs into GLCs by using BMP4 and RA.

Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs); where do they stand in tumorigenesis and how they can change the face of cancer therapy?

The tumor microenvironment (TME) and its components have recently attracted tremendous attention in cancer treatment strategies, as alongside the genetic and epigenetic alterations in tumor cells, TME could also provide a fertile background for malignant cells to survive and proliferate. Interestingly, TME plays a vital role in the mediation of cancer metastasis and drug resistance even against immunotherapeutic agents. Among different cells that are presenting in TME, tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) have shown to have significant value in the regulation of angiogenesis, tumor metastasis, and drug-resistance through manipulating the composition as well as the organization of extracellular matrix (ECM). Evidence has shown that the presence of both TAMs and CAFs in TME is associated with poor prognosis and failure of chemotherapeutic agents. It seems that these cells together with ECM form a shield around tumor cells to protect them from the toxic agents and even the adaptive arm of the immune system, which is responsible for tumor surveillance. Given this, targeting TAMs and CAFs seems to be an essential approach to potentiate the cytotoxic effects of anti-cancer agents, either conventional chemotherapeutic drugs or immunotherapies. In the present review, we aimed to take a deep look at the mechanobiology of CAFs and TAMs in tumor progression and to discuss the available therapeutic approaches for harnessing these cells in TME.

Performance evaluation of Fe-based water treatment sludge for dewatering of iron ore tailings slurry using coagulation-flocculation process: Optimization through response surface methodology.

This research attempted to investigate the feasibility of using drinking water treatment sludge (ferric chloride sludge, FCS) as a coagulant for turbidity removal from iron ore tailings slurry. The evaluation was performed in two phases. In the first phase, the one factor at a time (OFAT) approach was used to study the effects of FCS dosage, initial pH, and initial turbidity on turbidity removal efficiency (TR%) and the volume of the sediment produced at the end of the process (SV). In the second phase, response surface methodology (RSM) was employed to assess the individual and interaction effects of the parameters on TR% and SV. Numerical multiple-response optimization was carried out using RSM to maximize TR% and minimize SV simultaneously. At optimum condition (FCS dose of 0.13 g dried FCS/L, initial pH of 10, and initial turbidity of 538 NTU), the removal of all particles in the range of 0.25-1 µm and 2-55 µm from slurry led to the TR% of 78.80% and SV of 0.74 mL (per 250 mL of tailings). Characterization tests indicated that at alkaline pH values, the higher presence of hydroxide compounds intensified the enmeshment in a precipitate or sweep-floc mechanism, which was the predominant removal mechanism in this work. This study demonstrated the remarkable performance of FCS as a coagulant in water reclamation from iron beneficiation wastewater.

Release of heavy metals under pre-set redox potentials in Musa estuary sediments, northwestern of Persian Gulf.

Sediments are capable of adsorbing and desorbing heavy metals (HMs) under various environmental conditions. This study investigated the impact of pre-set redox potential (Eh) on the release dynamics of HMs (Co, Cr, Cu, Ni, Pb, V, and Zn) from sediment in an automated biogeochemical microcosm. The release of Co, Pb, and V under reducing conditions increased that may increase the potential risks in the aquatic environment. This phenomenon could be attributed to the decrease in pH, the reductive dissolution of FeMn oxides, and the complex of HMs with dissolved organic carbon (DOC). However, the soluble Cr, Cu, Ni, and Zn decreased at redox potentials as low as -150 mV. Co, Ni, Pb, and Zn were observed in mobile fractions while Cu primarily existed in the residual fraction (indicating lithogenic source). HPI and HEI indexes showed that water quality concerning HMs would become more unsuitable for aquatic life by reducing Eh.

Association of serum levels and receptor genes BsmI, TaqI and FokI polymorphisms of vitamin D with the severity of multiple sclerosis.

PURPOSE: Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease. Vitamin D has a major role in preventing inflammatory disorders. Therefore, any alteration in vitamin D receptor (VDR) might be a genetic risk factor for MS development. This study aimed to evaluate the effect of serum levels and VDR FokI, BsmI, and TaqI gene polymorphisms on the severity of MS. METHODS: This case-control study recruited 160 MS patients (71.9% females, mean age of 34.3 ± 8.3 years) and 162 (66.7% females, mean age 35.4 ± 7.9 year) age, sex, and ethnicity matched healthy controls. FokI (rs2228570), BsmI (rs1544410), and TaqI (rs731236) polymorphisms were carried out using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Demographic, clinical parameters, and the levels of vitamin D were compared between groups. RESULTS: We found that the frequency of FokI and TaqI polymorphisms significantly differed between the patients and the controls (p = 0.0127 and p = 0.0236, respectively). The MS patients had low levels of vitamin D compared to the controls (p = 0.011). In addition, TaqI T/C polymorphism significantly decreased the levels of vitamin D in the MS patients (p = 0.002). However, there was no significant association between FokI or BsmI SNPs and the levels of vitamin D in MS patients (p > 0.5). CONCLUSION: Our results suggest that FokI and TaqI polymorphisms of VDR are associated with MS risk and TaqI polymorphism is associated with Vitamin D levels in MS patients. Meanwhile, no difference was observed between VDR gene polymorphisms and any types of MS.

Fetal Cardiac Functional Abnormalities Assessed by Echocardiography in Mothers Suffering Gestational Diabetes Mellitus: A Systematic Review and Meta-analysis.

Abnormal cardiovascular changes especially hypertrophic cardiomyopathy is potentially expected in the fetuses of the diabetic pregnancy women. However, there is still little consensus on quantitative cardiac abnormalities in infants with diabetic mothers. The present study comprehensively analyzed the studies on functional changes in heart in infants of diabetic mothers with a greater focus on occurrence of hypertrophic cardiomyopathy. All comparative studies evaluating and comparing quantitatively the changes in cardiac parameters using echocardiography in fetuses with and without diabetic mothers were eligible for assessment. The included studies were identified through electronically reviewing the manuscripts databases of MEDLINE, EMBASE, Web of knowledge, and Google Scholar from inception to May 2020. The meta-analysis included 11 comparative with overall 849 fetuses for gestational diabetic mothers and 1247 for healthy mothers. Assessing cardiac diameters by fetal echocardiography showed significantly lower mitral E/A ratio, lower tricuspid E/A ratio, higher interventricular septal thickness, higher myocardial performance index, higher isovolumic relaxation time, and higher isovolumic contraction time in fetuses of gestational diabetes mellitus group as compared to healthy group adjusting for gestational diabetes mellitus. The presence of gestational diabetes mellitus can potentially affect the fetal cardiac parameters especially as hypertrophic cardiomyopathy leading both cardiac systolic and diastolic dysfunction.

Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR).

BACKGROUND: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). METHODS: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. RESULTS: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. CONCLUSION: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.

A comparative study of elemental pollution and health risk assessment in urban dust of different land-uses in Tehran's urban area.

Pollution levels of potentially toxic metals (PTMs) were studied performing enrichment factor, geo-accumulation index and ecological risk index. The most enriched elements which categorize as very high and extremely high enriched in different land-uses were in order of: commercial Cu (73.79)>Hg (66.55)>Pb (55.73)>Zn (46.15)>Cd (37.31); residential Pb (48.35)>Zn (34.79)>Cu (27.69)>Hg (22.96)>Cd (22.61) and industrial Zn (55.43)>Pb (51.52)>Cu (40.20)>Cd (26.29). In green lands sampling points, there weren't any elements with enrichment factor more than twenty. Based on the result of geo-accumulation index, commercial land-use appeared to be highly polluted with Cu, Pb and Zn. Residential land-use were highly polluted just in case of lead and industrial land-use were categorized as highly polluted in case of Zn, Pb and Cu. Hakanson's ecological risk index classified all of the land-uses except for green lands as very high ecological risk category. Factor analysis revealed that Cr, Mn, Ni, Fe and to a lesser extent Pb, Zn and As are from similar anthropogenic sources. Carcinogenic risk of Cr, Pb, As, Ni and Cd were found to be negligible for all land-uses for adult and children except for chromium in commercial land-use which exceed the safe level and need more attention in the future.

Contamination and cancer risk assessment of polycyclic aromatic hydrocarbons (PAHs) in urban dust from different land-uses in the most populated city of Iran.

Due to population growth and the considerable increase in usage of the resources, Human environment quality has been highly threatened by pollutants in recent decades. Polycyclic aromatic hydrocarbons (PAHs) are widespread, persistent organic pollutants which are of great concern due to their carcinogenicity. The present study is the first investigation that assesses contamination, sources and cancer risk of 16 priority PAHs proposed by US EPA in urban dust samples (n = 80) taken in different land-uses of Tehran metropolis, the capital of Iran. Gas chromatography-mass spectrometry (GC-MS) is used to measure PAHs concentrations. The results showed that the average concentration of the total 16 PAHs and the average Benzo[a]pyrene total potency equivalency were 566 µg kg-1 dry weight and 36.4 µg kg-1, respectively. In the commercial and residential land-uses high molecular weight (HMW) PAHs were dominated, whereas in green lands, light molecular weight (LMW) PAHs showed maximum contribution. The highest concentration of total PAHs were observed in the commercial areas due to limited air circulation and heavy traffic loads. Incremental Lifetime Cancer Risk (ILCR) model was applied to evaluate the cancer risk of exposure to PAHs contaminated dust. Based on the results, Tehran's residents (children and adults) in various land-uses except for green lands, are in high potential cancer risk of PAHs via ingestion and dermal contact exposure routs. Ace, Chr, Pyr, and BghiP which are indicators of traffic emissions, were found to be predominant PAH contributors in urban dust of commercial areas. Also, Ace, Fl, Phe, and BghiP which are derived from fossil fuel combustion, were mainly observed in the industrial land-use. Based on the results of factor analysis and diagnostic ratios, Diesel/gasoline engine vehicular emissions and combustion were found to be the main sources of PAHs in urban dust of Tehran.

Simultaneous removal of polycyclic aromatic hydrocarbons and heavy metals from natural soil by combined non-ionic surfactants and EDTA as extracting reagents: Laboratory column tests.

Simultaneous removal of three polycyclic aromatic hydrocarbons (acenaphthene, fluorene and fluoranthene) co-existing with three heavy metals (Ni, Pb and Zn) in artificially contaminated soil from the vicinity of an oil refinery was examined by column flushing of solutions containing Triton X-100 + Ethylenediaminetetraacetic acid (EDTA) and Tween 80 + EDTA at three levels of surfactant concentrations. While the effectiveness of both combined solutions in removal of heavy metals did not differ significantly, Triton X-100 + EDTA was more efficient in removing PAHs. Results showed that after 21 pore volume flushing of enhancing solution (Triton X-100 7.5% + EDTA 0.01 M) at flow rate of 0.534 mL min-1 through the column with hydraulic conductivity of 8.5 × 10-5 cm s-1, 54, 47 and 40% of acenaphthene, fluorene, and fluoranthene were removed simultaneously. At the same conditions, 75, 85 and 90% of Pb, Ni and Zn, were also simultaneously removed. Increasing the flow rate of flushing solution decreased the removal efficiency of the contaminants.

Micro-RNAs -106a and -362-3p in Peripheral Blood of Inflammatory Bowel Disease Patients.

OBJECTIVE: MicroRNAs (miRNAs) can regulate various genes after binding to target mRNAs. Studies on Inflammatory Bowel Disease (IBD) in relation with miRNA are much less shown. The aim of the present study was to assess the expression patterns of microRNA 106a and microRNA 362-3p in peripheral blood samples of Inflammatory Bowel Disease (IBD) patients including Crohn's Disease(CD) and Ulcerative Colitis (UC). METHODS: This study consisted of 32 CD, 32 UC patients and 32 controls. The expression level of the micro-RNAs -106a and -362-3p was determined using reverse transcription and real-time RT-PCR. RESULTS: Our findings showed that MiR-106a and miR-362-3p are expressed at significantly higher levels in the peripheral blood from patients with CD and UC compared to controls. MiR-106a and miR-362-3p expression are also different in the peripheral blood of patients regarding the activity score of the disease. There were significant differences of miR362-3p in active UC relative to inactive UC. CONCLUSION: Altogether our findings suggest that miR-106a and miR-363-3p can play an important role in the pathogenesis of IBD. The differences in expression of miR106a and miR362-3p in peripheral blood of the UC and CD patients in an active phase in comparison to inactive disease suggest that these miRNAs may be useful as potential biomarkers for diagnosis and monitoring the disease activity.

Desorption and mobility mechanisms of co-existing polycyclic aromatic hydrocarbons and heavy metals in clays and clay minerals.

The effects of soil components such as clay minerals and as humic acids, as well as co-existing metals and polycyclic aromatic hydrocarbons, on desorption and mobility are examined. Three types of artificially blended clay and clay mineral mixtures (pure kaolinite, kaolinite + sand and kaolinite + sand + bentonite), each with different humic acid content, were tested for desorption and mobility of acenaphthene, fluorene and fluoranthene by three extracting solutions CaCl2 (0.01 M) and EDTA (0.01M) with non-ionic surfactants (Tween 80 and Triton X100). Heavy metals (Ni, Pb and Zn) were also studied for desorption and mobility. The influence of co-present metals on simultaneous desorption and mobility of PAHs was investigated as well. The results showed that <10% of metals in the clay mineral mixtures were mobile. Combined EDTA and non-ionic solutions can enhance the desorption and mobility of PAHs to >80% in clay mineral mixtures containing no sand, while in the same soils containing ∼40% sand, the desorption exceeded 90%. Heavy metals, as well as increasing humic acids content in the clay mineral mixtures, decreased the desorption and mobility of PAHs, especially for soils containing no sand, and for fluoranthene compared with fluorene and acenaphthene.

Glucocorticoid-induced leucine zipper expression is associated with response to treatment and immunoregulation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder in which cytokine balance is disturbed. Glucocorticoids (GCs) are shown to balance immune response by transcriptional regulation of glucocorticoid receptor target genes such as Glucocorticoid-induced leucine zipper (GILZ) which has been introduced as an endogenous anti-inflammatory mediator. In the present study, we assessed the expression of GILZ in association with interferon-γ (IFN-γ), interleukine-10 (IL-10), and B lymphocyte stimulator (BLyS) plasma levels in SLE patients. A total of 40 female patients (18 under treatment and 22 newly diagnosed) were recruited in this study. Real-time RT PCR was conducted to quantify the mRNA expression of GILZ. The plasma levels of IFN-γ, IL-10, and BLyS were evaluated using ELISA method. GILZ was overexpressed among under treatment SLE patients. The mRNA expression of GILZ was significantly correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score. IFN-γ and BLyS were downregulated in response to therapies with negative correlations to GILZ. Moreover, IL-10 was upregulated among treated patients. The levels of IFN-γ and BLyS were correlated with the severity of disease, while IL-10 was negatively correlated with SLEDAI score. GILZ could be introduced as one of the acting molecules in mediating the regulatory effects of GCs on producing pro- and anti-inflammatory cytokines in SLE.

Multivariate analysis and geochemical approach for assessment of metal pollution state in sediment cores.

Vertical distribution of metals (Cu, Zn, Cr, Fe, Mn, Pb, Ni, Cd, and Li) in four sediment core samples (C1, C2, C3, and C4) from Anzali international wetland located southwest of the Caspian Sea was examined. Background concentration of each metal was calculated according to different statistical approaches. The results of multivariate statistical analysis showed that Fe and Mn might have significant role in the fate of Ni and Zn in sediment core samples. Different sediment quality indexes were utilized to assess metal pollution in sediment cores. Moreover, a new sediment quality index named aggregative toxicity index (ATI) based on sediment quality guidelines (SQGs) was developed to assess the degree of metal toxicity in an aggregative manner. The increasing pattern of metal pollution and their toxicity degree in upper layers of core samples indicated increasing effects of anthropogenic sources in the study area.

Fractionation of heavy metals in sediments and assessment of their availability risk: A case study in the northwestern of Persian Gulf.

Sequential extraction procedure was applied to determine the chemical forms of heavy metals in sediments in order to assess their mobility and availability in the aquatic environment. The mean concentrations of Cr, Ni, Pb, Cd, Cu and Zn in exchangeable and carbonate fractions were 14.9, 30.9, 17, 0.37, 4.4 and 14.4mg/kg, respectively. Based on the sediments quality guidelines (SQGs), the adverse biological effects caused by Ni, can occur frequently across the study area. Risk assessment code (RAC) suggested that the Cd has the highest level of environmental risk compared with other studied metals. Modified risk assessment code (mRAC) demonstrated a high potential adverse effect in the many of sampling sites. According to the fractionation results, the concentration of Ni in exchangeable and carbonate fractions was very considerable and more than other metals, hence, the potential risk of nickel release can be much more than other investigated metals.

Heavy metals relationship with water and size-fractionated sediments in rivers using canonical correlation analysis (CCA) case study, rivers of south western Caspian Sea.

Some pollutants can qualitatively affect aquatic freshwater such as rivers, and heavy metals are one of the most important pollutants in aquatic fresh waters. Heavy metals can be found in the form of components dissolved in these waters or in compounds with suspended particles and surface sediments. It can be said that heavy metals are in equilibrium between water and sediment. In this study, the amount of heavy metals is determined in water and different sizes of sediment. To obtain the relationship between heavy metals in water and size-fractionated sediments, a canonical correlation analysis (CCA) was utilized in rivers of the southwestern Caspian Sea. In this research, a case study was carried out on 18 sampling stations in nine rivers. In the first step, the concentrations of heavy metals (Cu, Zn, Cr, Fe, Mn, Pb, Ni, and Cd) were determined in water and size-fractionated sediment samples. Water sampling sites were classified by hierarchical cluster analysis (HCA) utilizing squared Euclidean distance with Ward's method. In addition, for interpreting the obtained results and the relationships between the concentration of heavy metals in the tested river water and sample sediments, canonical correlation analysis (CCA) was utilized. The rivers were grouped into two classes (those having no pollution and those having low pollution) based on the HCA results obtained for river water samples. CCA results found numerous relationships between rivers in Iran's Guilan province and their size-fractionated sediments samples. The heavy metals of sediments with 0.038 to 0.125 mm size in diameter are slightly correlated with those of water samples.