Development and Validation of a Fluorogenic Probe for Lysosomal Zinc Release.

Zinc homeostasis, which allows optimal zinc utilization in diverse life processes, is responsible for the general well-being of human beings. This paper describes developing and validating an easily accessible indole-containing zinc-specific probe in the cellular milieu. The probe was synthesized from readily available starting materials and was subjected to steady-state fluorescence studies. It showed selective sensing behavior towards Zn2+ with reversible binding. The suppression of PET (Photoinduced Electron Transfer) and ESIPT (Excited State Intramolecular Proton Transfer) elicited selectivity, and the detection limit was 0.63 µM (LOQ 6.8 µM). The zinc sensing capability of the probe was also screened in the presence of low molecular weight ligands [LMWLs] and showed interference only with GSH and ATP. It is non-toxic and can detect zinc in different cell lines under various stress conditions such as inflammation, hyperglycemia, and apoptosis. The probe could stain the early and late stages of apoptosis in PAN-2 cells by monitoring the zinc release. Most experiments were conducted without external zinc supplementation, showing its innate ability to detect zinc.

Potential probiotic Lactiplantibacillus plantarum strains alleviate TNF-α by regulating ADAM17 protein and ameliorate gut integrity through tight junction protein expression in in vitro model.

BACKGROUND: Lactiplantibacillus species are extensively studied for their ability to regulate host immune responses and functional therapeutic potentials. Nevertheless, there is a lack of understanding on the mechanisms of interactions with the hosts during immunoregulatory activities. METHODS: Two Lactiplantibacillus plantarum strains MKMB01 and MKMB02 were tested for probiotic potential following Indian Council of Medical Research (ICMR) guidelines. Human colorectal adenocarcinoma cells such as HT-29, caco-2, and human monocytic cell THP-1 were also used to study the potential of MKMB01 and MKMB02 in regulating the host immune response when challenged with enteric pathogen Salmonella enterica typhimurium. Cells were pre-treated with MKMB01 and MKMB02 for 4 h and then stimulated with Salmonella. qRT-PCR and ELISA were used to analyze the genes and protein expression. Confocal microscopy and field emission scanning electron microscopy (FESEM) were used to visualize the effects. An Agilent Seahorse XF analyzer was used to determine real-time mitochondrial functioning. RESULTS: Both probiotic strains could defend against Salmonella by maintaining gut integrity via expressing tight junction proteins (TJPs), MUC-2, and toll-like receptors (TLRs) negative regulators such as single Ig IL-1-related receptor (SIGIRR), toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase (IRAK)-M, A20, and anti-inflammatory transforming growth factor-ß and interleukin-10. Both strains also downregulated the expression of pro-inflammatory cytokines/chemokines interleukin-1ß, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), interleukin 6, and nitric oxide (NO). Moreover, TNF-α sheddase protein, a disintegrin and metalloproteinase domain 17 (ADAM17), and its regulator iRhom2 were downregulated by both strains. Moreover, the bacteria also ameliorated Salmonella-induced mitochondrial dysfunction by restoring bioenergetic profiles, such as non-mitochondrial respiration, spare respiratory capacity (SRC), basal respiration, adenosine triphosphate (ATP) production, and maximal respiration. CONCLUSIONS: MKMB01 and MKMB02 can reduce pathogen-induced gut-associated disorders and therefore should be further explored for their probiotic potential.

Harnessing Biomolecule-Infused 2D Multi-layered Luminescent Zn(II) Coordination Polymer for Electrochemical Energy Storage.

Selecting the right functional linkers and metal centers is crucial for creating multifunctional crystalline coordination polymers, which show promise in energy storage applications. Herein, a new two-dimensional Zn(II)-based CP, named BPHCC-1, has been synthesized using solvothermal methods with 2-amino terephthalic acid (2ATA) and the biomolecule purine as key building blocks. Purine, which is relatively unexplored in CP synthesis, plays a crucial role in the distinct properties of CPs. BPHCC-1, obtained as a stable crystalline solid, was characterized through various analytical techniques including Fourier transform infrared spectroscopy, field emission scanning electron microscope, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and Brunauer-Emmett-Teller analysis. The material's stability is attributed to extensive hydrogen bonding, π···π interactions, and coordination of the -NH2 group with the Zn(II) center. BPHCC-1 exhibits bright blue luminescence at 435 nm with a photoluminescence quantum yield of 29% in an aqueous dispersion. Furthermore, it demonstrates significant electrochemical energy storage performance, with a specific capacitance of 84 F g-1 at 3 A g-1 and retaining 64% of its original capacitance after 500 cycles. This study introduces a facile approach to designing multifunctional CPs, showcasing BPHCC-1's potential as a luminescent probe and pseudocapacitive supercapacitor. The findings highlight the versatility of BPHCC-1, suggesting broad opportunities for its use across diverse fields.

Heat-killed probiotic Levilactobacillus brevis MKAK9 and its exopolysaccharide promote longevity by modulating aging hallmarks and enhancing immune responses in Caenorhabditis elegans.

BACKGROUND: Proteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age-related diseases. RESULTS: This study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of-function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors. CONCLUSION: Our research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin-signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.

Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment.

The vaginal microenvironment of healthy women has a predominance of Lactobacillus crispatus, L. iners, L. gasseri, and L. jensenii. The genomic repertoire of the strains of each of the species associated with the key attributes thereby regulating a healthy vaginal environment needs a substantial understanding.We studied all available human strains of the four lactobacilli across different countries, isolated from vaginal and urinal sources through phylogenetic and pangenomic approaches. The findings showed that L. iners has the highest retention of core genes, and L. crispatus has more gene gain in the evolutionary stratum. Interestingly, L. gasseri and L. jensenii demonstrated major population-specific gene-cluster gain/loss associated with bacteriocin synthesis, iron chelating, adherence, zinc and ATP binding proteins, and hydrolase activity. Gene ontology enrichment analysis revealed that L. crispatus strains showed greater enrichment of functions related to plasma membrane integrity, biosurfactant, hydrogen peroxide synthesis, and iron sequestration as an ancestral derived core function, while bacteriocin and organic acid biosynthesis are strain-specific accessory enriched functions. L. jensenii showed greater enrichment of functions related to adherence, aggregation, and exopolysaccharide synthesis. Notably, the key functionalities are heterogeneously enriched in some specific strains of L. iners and L. gasseri.This study shed light on the genomic features and their variability that provides advantageous attributes to predominant vaginal Lactobacillus species maintaining vaginal homeostasis. These findings evoke the need to consider region-specific candidate strains of Lactobacillus to formulate prophylactic measures against vaginal dysbiosis for women's health.

Genome analysis of SARS-CoV-2 isolates from a population reveals the rapid selective sweep of a haplotype carrying many pre-existing and new mutations.

To understand the mechanism underlying the evolution of SARS-CoV-2 in a population, we sequenced 92 viral genomes from Assam, India. Analysis of these and database sequences revealed a complete selective sweep of a haplotype in Assam carrying 13 pre-existing variants, including a high leap in frequency of a variant on ORF8, which is involved in immune evasion. A comparative study between sequences of same lineage and similar time frames in and outside Assam showed that 10 of the 13 pre-existing variants had a frequency ranging from 96 to 99%, and the remaining 3 had a low frequency outside Assam. Using a phylogenetic approach to infer sequential occurrences of variants we found that the variant Phe120del on ORF8, which had a low frequency (1.75%) outside Assam, is at the base of the phylogenetic tree of variants and became totally fixed (100%) in Assam population. Based on this observation, we inferred that the variant on ORF8 had a selective advantage, so it carried the haplotype to reach the100% frequency. The haplotype also carried 32 pre-existing variants at a frequency from 1.00 to 80.00% outside Assam. Those of these variants that are more closely linked to the S-protein locus, which often carries advantageous mutations and is tightly linked to the ORF8 locus, retained higher frequencies, while the less tightly linked variants showed lower frequencies, likely due to recombination among co- circulating variants in Assam. The ratios of non-synonymous substitutions to synonymous substitutions suggested that some genes such as those coding for the S-protein and non-structural proteins underwent positive selection while others were subject to purifying selection during their evolution in Assam. Furthermore, we observed negative correlation of the Ct value of qRT-PCR of the patients with abundant ORF6 transcripts, suggesting that ORF6 can be used as a marker for estimating viral titer. In conclusion, our in-depth analysis of SARS-CoV-2 genomes in a regional population reveals the mechanism and dynamics of viral evolution.

Copper acetate catalysed C-C bond formation en route to the synthesis of spiro indanedione cyclopropylpyrazolones.

This article reports the synthesis of spiro compounds based on an indanedione-cyclopropane-pyrazolone framework. The reaction relied upon the Michael-initiated ring closure strategy and was carried out under Cu(OAc)2 catalysis, assisted by an oxygen atmosphere and the base Et3N. The final compounds were obtained as an inseparable mixture in most cases with modest to good yields using diverse substrates. Among the two plausible routes, computational studies indicated the feasibility of a route which involves a four-membered Cu containing intermediate. Given the generic nature of the developed method, it may be utilised to synthesise other analogous spiro systems.

Identification of the conserved long non-coding RNAs in myogenesis.

BACKGROUND: Our understanding of genome regulation is ever-evolving with the continuous discovery of new modes of gene regulation, and transcriptomic studies of mammalian genomes have revealed the presence of a considerable population of non-coding RNA molecules among the transcripts expressed. One such non-coding RNA molecule is long non-coding RNA (lncRNA). However, the function of lncRNAs in gene regulation is not well understood; moreover, finding conserved lncRNA across species is a challenging task. Therefore, we propose a novel approach to identify conserved lncRNAs and functionally annotate these molecules. RESULTS: In this study, we exploited existing myogenic transcriptome data and identified conserved lncRNAs in mice and humans. We identified the lncRNAs expressing differentially between the early and later stages of muscle development. Differential expression of these lncRNAs was confirmed experimentally in cultured mouse muscle C2C12 cells. We utilized the three-dimensional architecture of the genome and identified topologically associated domains for these lncRNAs. Additionally, we correlated the expression of genes in domains for functional annotation of these trans-lncRNAs in myogenesis. Using this approach, we identified conserved lncRNAs in myogenesis and functionally annotated them. CONCLUSIONS: With this novel approach, we identified the conserved lncRNAs in myogenesis in humans and mice and functionally annotated them. The method identified a large number of lncRNAs are involved in myogenesis. Further studies are required to investigate the reason for the conservation of the lncRNAs in human and mouse while their sequences are dissimilar. Our approach can be used to identify novel lncRNAs conserved in different species and functionally annotated them.

Iodine assisted synthesis of CF3 appended spirodihydrofuryl/cyclopropyl oxindoles by changing the active methylene sources.

This paper describes the synthesis of two distinct types of CF3-containing spirooxindoles by varying the active methylene sources. The reaction was carried out in DMSO, assisted by molecular iodine and Na2CO3via systematic application of Michael reaction and iodine mediated cyclisation. With 5-methyl-2,4-dihydro-3H-pyrazol-3-one as the methylene source, the final products obtained were spirodihydrofuropyrazolyl oxindoles, whereas 1H-indene-1,3(2H)-dione as the methylene source gave the final compounds spirocyclopropyl oxindoles. Modest to good yields were obtained for both the spiro systems.

Thickness-dependent magneto-transport properties of topologically nontrivial DyPdBi thin films.

DyPdBi (DPB) is a topological semimetal which belongs to the rare-earth-based half-Heusler alloy family. In this work, we studied the thickness-dependent structural and magneto-transport properties of DPB thin films (20 to 60 nm) grown using pulsed laser deposition. The DPB thin films show (110) oriented growth on MgO(100) single crystal substrates. Longitudinal resistance data indicate metallic surface states dominated carrier transport and the suppression of semiconducting bulk state carriers for films ≤40 nm. We observe the weak antilocalization (WAL) effect and Shubnikov-de Hass (SdH) oscillations in the magneto-transport data. The presence of a single coherent transport channel (α∼ -0.50) is observed in the Hikami-Larkin-Nagaoka (HLN) fitting of WAL data. The power law temperature dependence of phase coherence length (LØ ) ∼ T-0.50 indicates the observation of the 2D WAL effect and the presence of topological nontrivial surface states for films ≤40 nm. The 60 nm sample shows semiconducting resistivity behavior at higher temperature (>180 K) and HLN fitting results (α∼ -0.72, LØ âˆ¼ T-0.68 ) indicate the presence of partial decoupled top and bottom surface states. The Berry phase ∼π is extracted for thin films ≤40 nm, which further demonstrates the presence of Dirac fermions and nontrivial surface states. Band structure parameters are extracted by fitting SdH data to the standard Lifshitz-Kosevich formula. The sheet carrier concentration and cyclotron effective mass of carriers decrease with increasing thickness (20 nm to 60 nm) from ∼1.35 × 1012 cm-2 to 0.68 × 1012 cm-2 and from ∼0.26 me to 0.12 me, respectively. Our observations suggest that samples with a thickness ≤40 nm have transport properties dominated by surface states and samples with a thickness ≥60 nm have contributions from both bulk and surface states.

Selective Sensing of Iron by Pyrrolo[2,3-c]Quinolines.

This paper reports development of an iron sensor, 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (APQ). The fluorophore facilitates micromolar detection of Fe3+/Fe2+ in the presence of various cations, including well-known interfering cations Co2+and Cu2+ by the process of fluorescence quenching. Graphical Abstract.

Synthesis and anti-cancer activity of 1,4-disubstituted imidazo[4,5-c]quinolines.

The synthesis and anti-cancer activity evaluation of fused imidazoquinoline compounds is reported in this paper. Yb(OTf)3 has been utilized as a catalyst for the synthesis of 1,4-diaryl substituted imidazo[4,5-c]quinolines via a modified Pictet-Spengler approach. The desired imidazole ring was synthesized from imines using TosMIC (toluenesulfonylmethyl isocyanide) and subsequently functionalized at the C-4 position yielding an imidazoquinoline skeleton. Importantly, the final step was carried out without the aid of any prefunctionalization to obtain the resultant compounds in good yields. The synthesized compounds, when screened for anti-cancer activity, revealed the highest activity with 4-(2-bromophenyl)-1-phenyl-1H-imidazo[4,5-c]quinoline (IC50: 103.3 µM).

Synthesis of 4-substituted oxazolo[4,5-c]quinolines by direct reaction at the C-4 position of oxazoles.

A facile synthesis of 4-aryl substituted oxazolo[4,5-c]quinolines has been described via a modified Pictet-Spengler method and using Cu(TFA)2 as a catalyst. The developed methodology directly functionalizes the C-4 position of oxazoles without the aid of any prefunctionalization, in the presence of the more reactive C-2 position in good yields. The versatility of the established method has been demonstrated by its application in the synthesis of 4-substituted oxazolo-[1,8]naphthyridine ring systems.

Understanding selective sensing of human serum albumin using a D-π-A probe: a photophysical and computational approach.

The human serum albumin (HSA) level is a valuable indicator of an individual's health status. Therefore, its detection/estimation can be used to diagnose several diseases. In this work, we have developed a series of donor-π-acceptor probes, which were found to selectively detect HSA over BSA (bovine serum albumin). Among these probes, A4, which bears the trifluoroacetyl group, showed the highest selectivity for HSA, with limits of detection and quantification being 1.36 nM and 2.59 nM, respectively. CD spectroscopy of the HSA-A4 ensemble indicated an increase in the α-helicity of the protein, while the displacement assays revealed the localization of the probe in the hemin site of HSA. The probe works on the principle of excited state intramolecular charge transfer (ICT). Its selectivity was also validated computationally. Docking experiments confirmed the preference of the probe for the hemin binding IB site of HSA, as observed from the fluorescence displacement assay results, and a comparison of docking scores demonstrated the greater preference of A4 for HSA compared to BSA. Computational experiments also showed a change in preference for HSA amino acid residues exhibited by the excited state of probe A4 (Tyr161, Met123, Pro118, and Leu115) when compared to its ground state (Arg186 and His146). Hydrophobic interactions dominated the excited state protein-probe ensemble, whereas there was significant involvement of the water bridges along with the hydrophobic interactions in the ground state ensemble. Probe A4 was also assessed for its practical utility and found to successfully sense HSA in urine at extremely low concentrations. Moreover, the A4-HSA ensemble was employed for hemin sensing with a detection limit of 0.23 µM.

HSA over BSA: Selective detection of Human Serum Albumin via a naphtho [2,1-b] furan-based system.

Human serum albumin (HSA) is an important biomarker that can be used for the early diagnosis of many diseases. In this work, a TICT probe bearing fused naphtho-furan scaffold (NPNF) was developed and employed in the selective turn-on sensing of HSA. The probe's selectivity towards HSA was observed using steady-state fluorescence experiments, with limit of quantitation in micromolar levels. NPNF's capability to exclusively detect HSA over BSA was further studied/rationalized using anisotropy and time-resolved studies. Molecular docking was used to shed light on the location of NPNF in the subdomain IB of HSA. The practical application of the probe was also demonstrated by the detection of HSA in urine and the HSA-assisted detection of cerium.

Probiotic and anti-inflammatory properties of Lactiplantibacillus plantarum MKTJ24 isolated from an artisanal fermented fish of North-east India.

The study aimed to isolate and characterize lactic acid bacteria from various traditional fermented fish products from North East India, including Xindol, Hentak, and Ngari, which hold significant dietary importance for the indigenous tribes. Additionally, the study sought to examine their untargeted metabolomic profiles. A total of 43 strains of Bacillus, Priestia, Staphylococcus, Pediococcus, and Lactiplantibacillus were isolated, characterized by 16 S rRNA gene and tested for probiotic properties. Five strains passed pH and bile salt tests with strain dependent antimicrobial activity, which exhibited moderate autoaggregation and hydrophobicity properties. Lactiplantibacillus plantarum MKTJ24 exhibited the highest hydrophobicity (42 %), which was further confirmed by adhesion assay in HT-29 cell lines (100 %). Lactiplantibacillus plantarum MKTJ24 treatment in LPS-stimulated HT-29 cells up-regulated expression of mucin genes compared to LPS-treated cells. Treatment of RAW 264.7 cells with Lactiplantibacillus plantarum MKTJ24 decreased LPS-induced reactive oxygen species (ROS) and nitric oxide (NO) productions. Further, genome analysis of Lactiplantibacillus plantarum MKTJ24 revealed the presence of several probiotic markers and immunomodulatory genes. The genome was found to harbor plantaricin operon involved in bacteriocin production. A pangenome analysis using all the publicly available L. plantarum genomes specifically isolated from fermented fish products identified 120 unique genes in Lactiplantibacillus plantarum MKTJ24. Metabolomic analysis indicated dominance of ascorbic acids, pentafluropropionate, cyclopropaneacetic acid, florobenzylamine, and furanone in Xindol. This study suggests that Lactiplantibacillus plantarum MKTJ24 has potential probiotic and immunomodulatory properties that could be used in processing traditional fermented fish products on an industrial scale to improve their quality and enhance functional properties.

To Quench or Not: Extending a ß-Carboline Fluorophore for TNT Detection in Aqueous Media via Simultaneous ESPT Destabilization and AIE.

Molecules relying on the excited-state intramolecular proton transfer/excited-state proton transfer (ESIPT/ESPT) mechanism are widely used in material science. In the current work, a known ß-carboline-based probe TrySy was used to selectively detect explosive trinitrotoluene (TNT) in water. Compared to conventional TNT sensing, which relies mainly on the quenching of the fluorescence signal, TrySy could perform nanomolar detection of TNT via ESPT destabilization and AIE, with a significant fluorescence output. The mechanism followed was validated by computational and experimental results.

Visible light sensing of ions by a cyanoquinoxaline 1,4-dioxide-based probe and its applications.

Inducting newer fluorophores for colourimetry/fluorimetry-assisted analyte sensing is of great importance. Towards this end, we have shown the application of quinoxaline-1,4-dioxide bioactive molecules for the first time as potential probes for cations and anions. The molecule (ACQ) used in this study is soluble in water and provides specific colour output upon interaction with copper and palladium ions. Changing the solvent to DMSO allows a change in selectivity to fluoride ions via pink to blue colour change. All the ions detected showed quenching of the fluorescence signal upon interaction with the probe. Analysis of the Stern-Volmer plot indicated the predominant role of static quenching in the selective ion-sensing behaviour of the probe. The stoichiometry of the ACQ and ion was 2 : 1 in the case of Cu2+ and Pd2+, whereas a ratio of 1 : 1 was seen in the case of F-. We have also applied ACQ to probe the above-mentioned analytes in practical settings.

Multi-Omics Analysis Demonstrates the Critical Role of Non-Ethanolic Components of Alcoholic Beverages in the Host Microbiome and Metabolome: A Human- and Animal-Based Study.

It is known that alcoholic beverages alter the human gut microbiome. This study focused on the potential impact of non-ethanolic ingredients in whisky on the gut bacteriome. A pilot study was carried out on 15 whisky drinkers, 5 rice beer drinkers, and 9 non-drinkers to determine the effect of alcoholic beverages on the host microbiome and metabolome. Additionally, a mouse model was used to assess the differential impact of three whisky brands (each with an equal ethanol concentration). The results indicate that the non-ethanolic components have an impact on the gut microbiome, as well as on the metabolites in blood and feces. The amount of Prevotella copri, a typical core Indian gut bacterium, decreased in both the human and mouse groups of whisky type 1, but an increase in abundance of Helicobacteriaceae (p = 0.01) was noticed in both groups. Additionally, the alcohol-treated cohorts had lower levels of short-chain fatty acids (SCFAs), specifically butyric acid, and higher amounts of lipids and stress marker IL1-ß than the untreated groups (p = 0.04-0.01). Furthermore, two compounds, ethanal/acetaldehyde (found in all the whisky samples) and arabitol (unique to whisky type 1), were tested in the mice. Similar to the human subjects, the whisky type 1 treated mouse cohort and the arabitol-treated group showed decreased levels of Prevotella copri (p = 0.01) in their gut. The results showed that non-ethanolic compounds have a significant impact on host gut bacterial diversity and metabolite composition, which has a further vital impact on host health. Our work further emphasizes the need to study the impact of non-ethanolic ingredients of alcoholic beverages on host health.