Synergistic impact of bioavailable PHEs and alkalinity on microbial diversity and traits in agricultural soil adjacent to chromium-asbestos mines.

Soil microbial communities undergo constant fluctuations, particularly in response to environmental factors. Although the deposition of toxic mine waste is recognized for introducing potentially hazardous elements (PHEs) into the soil, its specific impacts on microbial communities remain unclear. This study aims to explore the combined effects of soil alkalinity and bioavailable PHEs on microbial diversity and traits in agricultural soil adjacent to a chromium-asbestos mining area. By employing a comprehensive analysis, this study indicated that microbiological attributes were reduced in contaminated areas (zone 1), whereas both the levels of bioavailable PHEs (CrWs: 31.08 mg/kg, NiWs: 13.90 mg/kg) and alkalinity indices (CROSS, MCAR, MH) were significantly higher. The spatial distribution of soil alkalinity and bioavailable PHEs, primarily originating from chromium-asbestos mines, has been determined. This study also elucidates the negative relationship between soil stressors (Alkalinity and PHEs) and microbial activities (soil enzymatic activity, microbial respiration, and biomass carbon). The vector's length exhibited a notable difference between zone 1 (0.51) and zone 2 (0.32), indicating a substantial limitation on carbon (C). Also, the investigation of soil bacterial diversity unveiled notable disparities in the prevalence of microbial populations inside zone 1. Proteobacteria constituted 57.18% of the total population indicating a noteworthy prevalence in the contaminated soils. Finally, the random forest (RF) algorithm from machine learning was selected and proven to be a robust choice in Taylor diagrams for predicting the causative stressors responsible for the deterioration of soil microbial health. Therefore, this research offers insights into the health and resilience of soil microbial communities under synergistic stress conditions, which will aid environmentalists in planning future interventions and improving sustainable farming techniques.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

Chemical and toxicological studies on black crust formed over historical monuments as a probable health hazard.

Studies to date have mostly investigated environmental factors responsible for deterioration of historical monuments. Black crusts formed on historical monuments are considered as factor for deterioration of structures or as an indicator of environmental status of the surrounding area. Black crust formed on historical monuments has never been investigated as a health hazard. Herein, for the first time, we performed in vitro and in vivo toxicology studies of black crust formed on three culturally-rich historical monuments (Rang Ghar, Kareng Ghar, and Talatal Ghar) of the Indian subcontinent to test their toxicological effect. Black crust suspension in ultrapure water was found not to be considerably toxic to the cells upon direct short-term exposure. However, the sub-acute nasal exposure of the black crust suspension in Swiss albino mice produced lung-specific pathologies and mortality. Additionally, structural formation of the black crust along with the speciation of potentially hazardous elements (PHEs), polyaromatic hydrocarbon (PAHs), and other metals were investigated. Overall, these results indicate the potential of black crust deposited on historical monuments as health hazard owing to the atmospheric pollution of the surroundings. However, it may be noted that black crust and its components have very low possibility of health implication unless they are disturbed without proper care.

Performance of EncephalApp Stroop Test in Cirrhotic Patients for Evaluating Covert Hepatic Encephalopathy.

BACKGROUND: Covert hepatic encephalopathy (HE) is the mildest HE spectrum that is difficult to detect, but associated with significant decrease in quality of life. Currently, there is no gold standard to detect covert HE. EncephalApp Stroop Test as a newer diagnostic tool is easier, faster and its ease of availability in various health institutions is expected to be applied in Indonesia for covert HE detection. This study aimed to validate and test the reliability and diagnostic ability of EncephalApp Stroop Test to diagnose covert HE, compared to the Psychometric Hepatic Encephalopathy Score (PHES) and critical flicker frequency (CFF). METHODS: This study is a cross-sectional test, conducted from August to September 2018, targeted at patient with cirrhosis in Jakarta, to obtain Area Under The Curve (AUC), sensitivity, specificity, cut-off point, predictive value, likelihood ratio, and post-test probability of the EncephalApp Stroop Test, compared to PHES and CFF. The Validity and reliability tests were done before diagnostic study. Translation of the EncephalApp Stroop Test were first carried out using WHO protocol. All patients first underwent a Mini Mental State Examination and Ishihara Test to rule out color blindness. RESULTS: Thirty subjects participated in validity and reliability tests, and eighty in diagnostic tests. The translated application showed excellent internal consistency (Chronbach's Alpha of 0.942) and correlation coefficient of 0.82. The diagnostic study showed OnTime + OffTime as the best parameter (AUC: 0.897 (95% CI: 82.9% - 96.5%); sensitivity: 88.6%; specificity: 80%; positive predictive value (PPV): 0.77; negative predictive value (NPV): 0.9; positive likelihood ratio (LK+): 4.4; negative likelihood ratio (LK-): 1.4; positive post-test probability: 0,775; negative post-test probability: 0,1; and cut-off point ≥ 188.8 seconds. CONCLUSION: The EncephalApp Stroop Test is valid and reliable, with good AUC value, sensitivity, specificity, PPV, NPV and likelihood ratio in diagnosing covert hepatic encephalopathy in patients with cirrhosis in Indonesia.

Animal naming test is a simple and valid tool for detecting covert hepatic encephalopathy and predicting outcomes in Chinese-speaking regions: a preliminary study.

BACKGROUND AND AIMS: Hepatic encephalopathy (HE) implies high morbidity and mortality. The assessment of covert HE (CHE) [i.e. minimal HE (MHE) plus grade 1 HE] is often neglected in Taiwan. Therefore, the aim was to investigate the potential of the animal naming test (ANT1 and simplified ANT1 (S-ANT1)) for assessing CHE in Chinese-speaking regions, specifically Taiwan. METHODS: A prospective cohort study was conducted, comprising 65 cirrhotic patients and 29 healthy controls (relatives of the patients). Patients were followed up every three months and censored after two years or until death. Hospitalization for overt HE (OHE) and mortality were considered. All subjects underwent ANT1, psychometric HE score (PHES), and mini-mental state examination (MMSE). The patients underwent an electroencephalogram (EEG) to detect slowing indicative of MHE. Cut-off values for ANT1 and S-ANT1 were assessed by ROC analysis and Youden's index, considering CHE as a reference. The prognostic values for OHE and OHE-free survival were assessed. RESULTS: Preliminary analysis confirmed that PHES ≤-4 is a good discriminant point for abnormal results. CHE was found in 29 patients: 9 had MHE (PHES ≤ -4 or altered EEG) and 20 had grade 1 HE. ANT1 and S-ANT1 were found to have diagnostic values for CHE: AUC = 0.807, 0.786; cut off: 18 and 19, respectively. ANT1 and S-ANT1 were found to have prognostic value for OHE, number of hospitalization episodes for OHE, and OHE recurrence-free survival. CONCLUSIONS: ANT1 shows promise as a tool for CHE detection, quantification, and follow-up in Taiwan and other Chinese-speaking regions.Key messagesThe animal naming test (ANT1) is a simple and valid semantic fluency test that can be easily performed in outpatient or bedside settings in one minute and can also be used as a tool for covert hepatic encephalopathy (CHE) detection, quantification, and follow-up in Taiwan, other Chinese-speaking regions, and many other countries.The diagnostic value of ANT1 and S-ANT1 for CHE were found to be significant, with area under the receiver operating characteristic curve (AUROC) values of 0.807 and 0.786 respectively, and cut-off scores of 18 and 19.ANT1 and S-ANT1 have prognostic value for the first breakthrough of overt hepatic encephalopathy (OHE), number of hospitalization episodes for OHE, and OHE recurrence-free survival, independent of the MELD score.

Elevated serum levels of glial fibrillary acidic protein are associated with covert hepatic encephalopathy in patients with cirrhosis.

Background & Aims: Blood biomarkers facilitating the diagnosis of covert hepatic encephalopathy (CHE) in patients with cirrhosis are lacking. Astrocyte swelling is a major component of hepatic encephalopathy. Thus, we hypothesised that glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, might facilitate early diagnosis and management. This study aimed to investigate the utility of serum GFAP (sGFAP) levels as a biomarker of CHE. Methods: In this bicentric study, 135 patients with cirrhosis, 21 patients with ongoing harmful alcohol use and cirrhosis, and 15 healthy controls were recruited. CHE was diagnosed using psychometric hepatic encephalopathy score. sGFAP levels were measured using a highly sensitive single-molecule array (SiMoA) immunoassay. Results: In total, 50 (37%) people presented with CHE at study inclusion. Participants with CHE displayed significantly higher sGFAP levels than those without CHE (median sGFAP, 163 pg/ml [IQR 136; 268] vs. 106 pg/ml [IQR 75; 153]; p <0.001) or healthy controls (p <0.001). sGFAP correlated with results in psychometric hepatic encephalopathy score (Spearman's ρ = -0.326, p <0.001), model for end-stage liver disease score (Spearman's ρ = 0.253, p = 0.003), ammonia (Spearman's ρ = 0.453, p = 0.002), and IL-6 serum levels (Spearman's ρ = 0.323, p = 0.006). Additionally, sGFAP levels were independently associated with the presence of CHE in multivariable logistic regression analysis (odds ratio 1.009; 95% CI 1.004-1.015; p <0.001). sGFAP levels did not differ between patients with alcohol-related cirrhosis vs. patients with non-alcohol-related cirrhosis or between patients with ongoing alcohol use vs. patients with discontinued alcohol use.Conclusions: sGFAP levels are associated with CHE in patients with cirrhosis. These results suggest that astrocyte injury may already occur in patients with cirrhosis and subclinical cognitive deficits and that sGFAP could be explored as a novel biomarker. Impact and implications: Blood biomarkers facilitating the diagnosis of covert hepatic encephalopathy (CHE) in patients with cirrhosis are lacking. In this study, we were able to demonstrate that sGFAP levels are associated with CHE in patients with cirrhosis. These results suggest that astrocyte injury may already occur in patients with cirrhosis and subclinical cognitive deficits and that sGFAP could be explored as a novel biomarker.

Heavy metal contamination levels, source distribution, and risk assessment in fine sand of urban surface deposited sediments of Ekaterinburg, Russia.

Urban surface deposited sediments (USDS) are unique indicators of local pollution that pose a potential threat to the living environment and human health. Ekaterinburg is a highly populated metropolitan area in Russia with rapid urbanization and industrialization activities. In Ekaterinburg's residential areas, about 35, 12, and 16 samples are represented by green zones, roads, driveways, and sidewalks, respectively. The total concentrations of heavy metals was detected using a chemical analyzer inductively coupled plasma mass spectrometry (ICP-MS). Zn, Sn, Sb, and Pb have the highest concentrations in the green zone, while V, Fe, Co, and Cu represent the utmost values on roads. Moreover, Mn and Ni are the prevailing metals in the fine sand fraction of driveways along with sidewalks. Broadly, the high pollution in the studied zones is generated by anthropogenic activities and traffic emissions. The potential ecological risk (RI) was observed in high risk (IR > 600), even though the results of all heavy metals reveal no adverse health effects from the considered noncarcinogenic metal for adults and children by different exposure pathways except the children's exposure to Co in case of the dermal contact, where the HI values of Co for children in the studied zones are higher than the proposed level (> 1). In all urban zones, the total carcinogenic risk (TLCR) values are predicted as a high potential inhalation exposure.

Evolution of hepatic encephalopathy over time: ecological data from a tertiary referral centre for hepatology.

OBJECTIVES: Few data on hepatic encephalopathy (HE) over time are available, thus our aim was to study its evolution in patients with varying degree of HE on first assessment. METHODS: Eighty-six patients with cirrhosis (age = 58 ± 11 years; males = 72) were evaluated 2-10 times for liver transplantation selection purposes, differential diagnosis or treatment optimization. The presence/severity of HE was assessed by clinical and neuropsychiatric indices [ Psychometric Hepatic Encephalopathy Score (PHES) and electroencephalography (EEG)] and the severity of liver disease by the Model for End-Stage Liver Disease (MELD) score. Treatment was instituted/modified after each evaluation. RESULTS: Amongst 23 unimpaired patients, 56/6% remained unimpaired, 35/3% developed covert HE, 9/0% developed overt HE on second/third evaluation. Amongst 32 patients with covert HE, 25/10% became unimpaired, 44/19% remained covert, 31/13% developed overt HE. Finally, amongst 32 patients with overt HE, 19/16% became unimpaired, 25/13 % became covert and 56/25% remained overt. PHES results improved in patients with overt HE and EEG worsened over time (despite remaining normal) in unimpaired patients. In patients with multiple evaluations, HE evolution was manifold and difficult to predict. CONCLUSIONS: HE evolution over time is variable and largely dependent on HE history/management. These data support the concept that HE is an essentially reversible condition.

PMA-qPCR method for the selective quantitation of viable lactic acid bacteria in fermented milk.

The number of viable lactic acid bacteria (LAB) is a key indicator of the quality of fermented milk. Currently, the combination of propidium monoazide (PMA) and qPCR has been applied in the quantification of viable bacteria in various matrices. In this research, the PMA-qPCR method was used to detect the number of viable bacteria of each LAB species in fermented milk. By analyzing pheS gene and 16S rRNA gene sequence similarities in five species of LAB, namely Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, the pheS gene resolved species identities better and was thus selected to design specific primers and probes. The pheS gene was cloned into the pUC19 vector and used to construct a standard curve for absolute quantification. Standard curves for quantification were constructed for each LAB species for serial dilutions between 1011 and 106 CFU/mL, with R 2 > 0.99. The number of viable bacteria in the fermented milk detected by PMA-qPCR was significantly lower than that of qPCR (P < 0.05), indicating that PMA inhibited the amplification of DNA from dead cells. This was corroborated by the results from bacterial staining and plate count experiments. The proposed PMA-qPCR method provided rapid qualitative and quantitative determination of the number of viable bacteria for each LAB species in fermented milk within 3 h.

Highly Effective Markerless Genetic Manipulation of Streptococcus suis Using a Mutated PheS-Based Counterselectable Marker.

Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.