2019-2020 Bushfire impacts on sediment and contaminant transport following rainfall in the Upper Murray River catchment.

During the 2019-2020 Australian bushfire season, large expanses (~47%) of agricultural and forested land in the Upper Murray River catchment of southeastern (SE) Australia were burned. Storm activity and rainfall following the fires increased sediment loads in rivers, resulting in localized fish kills and widespread water-quality deterioration. We collected water samples from the headwaters of the Murray River for sediment and contaminant analysis and assessed changes in water quality using long-term monitoring data. A robust runoff routing model was used to estimate the effect of fire on sediment loads in the Murray River. Peak turbidity in the Murray River reached values of up to 4200 nephelometric turbidity units (NTU), shown as pitch-black water coming down the river. The increase in suspended solids was accompanied by elevated nutrient concentrations during post-bushfire runoff events. The model simulations demonstrated that the sediment load could be five times greater in the first year after a bushfire than in the prefire condition. It was estimated that Lake Hume, a large reservoir downstream from fire-affected areas, would receive a maximum of 600 000 metric tonnes of sediment per month in the period immediately following the bushfire, depending on rainfall. Total zinc, arsenic, chromium, nickel, copper, and lead concentrations were above the 99% toxicant default guideline values (DGVs) for freshwater ecosystems. It is also likely that increased nutrient loads in Lake Hume will have ongoing implications for algal dynamics, in both the lake and the Murray River downstream. Information from this study provides a valuable basis for future research to support bushfire-related policy developments in fire-prone catchments and the mitigation of postfire water quality and aquatic ecosystem impacts. Integr Environ Assess Manag 2021;17:1203-1214. © 2021 Commonwealth of Australia. Integrated Environmental Assessment and Management © 2021 Society of Environmental Toxicology & Chemistry (SETAC).

Identification and expression profiling of MSY genes of yak for bull fertility.

Yak (Bos grunniens) is a unique bovine species and considered as lifeline of highlanders. The male subfertility in yak is a matter of concern that causes huge economic loses. The spermatogenesis and male reproduction machinery are critically governed by Y-linked genes which tend to acquire necessary information in the course of evolution. The Y-linked fertility genes are present in multiple copies with testis-limited expression. To understand this novel complexity, 12 male-specific region of Y chromosome (MSY) genes have been studied in the yak. Targeted genes are amplified in male and female genomic DNA and confirmed the male derived specificity. Moreover, testis and sperm-specific expressions of MSY genes are distinct among different tissues. The quantitative polymerase chain reaction results validate the expression pattern of these genes in various tissues with predominant expression intestis and sperm. The sequencing of resultant yak MSY genes gives significant result and shows similarity with cattle (Bos indicus), but few nucleotide mismatches define the proposition of infertile male in the F1 hybrid of cattle and yak. The identified MSY genes can be used to establish male-specific characteristics and to differentiate male and female yak genotypically. Further, these genes may act as valuable resources to understand the capacity of spermatogenesis, embryogenesis, cellular growth, azoospermia and malesubfertility in the yak.

Fate and dynamics of metal precipitates arising from acid drainage discharges to a river system.

Neutralisation of acid drainage creates metal-rich precipitates that may impact receiving water bodies. This study assessed the fate of over seven years of acid drainage discharges on the sediments of the lower River Murray (Australia), including the potential for periodic water anoxia to enhance risk via reductive dissolution of amorphous (Fe, Mn and co-precipitated and bound metal) oxide phases. With the exception of one site with restricted water exchange, elevated reducible/reactive metal(oid) (Fe, Ni, As, Co, Zn) concentrations were only observed in the localised wetland-riparian area within approximately 100 m of the discharges. Only a minor exceedance of national sediment quality guideline values occurred for Ni. In the main river channel, elevated reactive metal (Fe, Mn, Ni, Zn) concentrations were also only observed less than approximately 100 m from the drainage discharge point. This appears due to (a) rapid neutralisation of pH leading to metal precipitation and deposition in the localised discharge area, and/or (b) dilution of any metal precipitates entering the main channel with natural river sediments, and/or (c) flushing of precipitates downstream during higher flow conditions. The influence of deoxygenation on metal release was profound with large increases in the concentration of dissolved Fe, Mn, Zn, Ni, and As in the overlying water during laboratory experimental simulations. However, given in situ sediment metal contamination is very localised, it appears on a river reach scale that the acid drainage precipitates will not significantly contribute, over and above, the background release of these metals during these conditions.

Prevalence of paratuberculosis in organized and unorganized dairy cattle herds in West Bengal, India.

AIM: The aim of this study was to determine the prevalence pattern of Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis or Johne's disease, in unorganized as well as organized cattle herds in West Bengal. MATERIALS AND METHODS: Four organized cattle farms with identical management practice in Nadia (n=3) and South 24 Parganas (n=1) districts and three unorganized cattle herds, one each from three districts, namely, Burdwan, North 24 Parganas, and Purba Midnapur, were selected randomly and screened for paratuberculosis by delayed-type hypersensitivity (DTH) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 191 animals tested by DTH, 57 (29.8%) were found to be positive in comparison to 72 (37.7%) by ELISA. In organized farms, seropositivity varied from 13.3% to 53.1%, whereas in unorganized sector, it ranged from 5% to 6.7% with one area having exceptionally high prevalence, i.e. 53.3%. The range of positivity detected by DTH both in organized farms and backyard sectors varied from 0% to 46.7%. By employing both DTH and ELISA together, the positivity of animals in organized and unorganized herds was 19.9% and 8%, respectively. CONCLUSION: The results indicate that animals in organized farms are much more prone to paratuberculosis than others. For screening the herd, both DTH and ELISA should be used simultaneously to increase the test sensitivity in order to minimize its further spread adopting control programs.

IL-15 stimulates NKG2D while promoting IgM expression of B-1a cells.

Interleukin (IL)-15, a key manipulator of T-cell function also modulates B-1a cell activity by augmenting activation markers, turning them towards type 1 polarization and immunoglobulin (Ig) expression which is significant in the context of gut immunity. Here we show, for the first time, IL-15 mediated up-regulation of the activation receptor NKG2D and its adaptor DAP10 in B-1a cells indicating their essential coupling with IL-15 receptor signaling pathway. Our results demonstrate IL-15 treatment increases phosphorylation of STAT5 and p38 leading to translocation of NF-κB onto the nucleus, an attribute that delineates activation of B-1a cells and its role in inflammation. In parallel, increase of anti-apoptotic Bcl-xL suggests its role in long term survival of B-1a cells in culture by IL-15. The cytokine induced overexpression of the plasma cell differentiation transcription factor BLIMP-1 while reducing PAX-5a that could be responsible for the spontaneous Ig secretion by B-1a cells. Up-regulation of IgM transcripts in presence of IL-15 validates mucosal response of the cells through natural Abs to counter pathogens.

Pathogen-associated porin turns IL-10 competent B-1a cells toward proinflammatory cytokine response.

Shigellosis is a major problem in the developing countries causing mortality and morbidity particularly among the children. Shigella spp. harbours the epithelial cells of the human colon to infect the host and spread the disease. We analyzed the response of B-1a cells, the major component of the mucosal immune system to porin of Shigella dysenteriae type 1. We show that porin while proliferating B-1a cells, deplete Siglec-G, the inhibitory molecule present on B-1a cells. Adjuvanticity of porin has been shown to govern innate signaling for promoting host adaptive immune response. Up-regulation of CD69 and CD40 denotes activation of the cells parallel to abrogation of Siglec-G. As a result of cell activation, porin stimulated the inflammatory cytokines of CD5+ B-1a cells, otherwise rich in IL-10. The work shows B-1a cell responses promote the immunopotentiating activity of porin.

IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression.

Interleukin (IL)-15 is known to strongly modulate T-cell function; however, its role in controlling mucosal immunity, including its ability to modulate B-1a cell activity, remains to be elucidated. Here, we show that IL-15 upregulates activation molecules and the costimulatory molecule CD80 on viable B-1a cells. Cell activation was accompanied by the depletion of sialic acid-binding immunoglobulin-like lectin (Siglec)-G, an inhibitor of cell activation that is present on B-1a cells. The IL-15 receptor CD122 was stimulated on B-1a cells by the cytokine showing its direct involvement in IL-15-mediated responses. IL-10 is responsible for the long term survival of B-1a cells in culture, which is initially promoted by IL-15. The upregulation of IL-10 was followed by the appearance of suppressor of cytokine signaling (SOCS)1 in the presence of IL-15 and the loss of IL-10. This resulted in the cells switching to IL-12 expression. This anti-inflammatory to pro-inflammatory shift in the B-1a cell character was independent of the cell-specific marker CD5, which remained highly expressed throughout the in vitro life of the cells. The presence of the immunosuppressive receptor programmed cell death (PD)-1 and its ligand PD-L2 were features of a predominantly IL-10 response. PD-1 and PD-L2 can mediate juxtacrine signaling. However, the abrogation of PD-1 and its ligand was observed when the cells expressed IL-12. This demonstrates an inverse relationship between the receptor and ligand and the pro-inflammatory cytokine. The induction of IgM and IgA, which can play pivotal roles in mucosal immunity, was promoted in the presence of IL-15. Collectively, the data implicate IL-15 as the master cytokine that induces B-1a cells to mount a mucosal immune response.

Enthalpy-Entropy Compensation (EEC) Effect: A Revisit.

A short account of the developments and perspectives of IKR (iso-kinetic relation) and EEC (enthalpy (H) - entropy (S) compensation) has been presented. The IKR and EEC are known to be extra thermodynamic or empirical correlations though linear H-S correlation can be thermodynamically deduced. Attempt has also been made to explain the phenomena in terms of statistical thermodynamics. In this study, we have briefly revisited the fundamentals of both IKR and EEC from kinetic and thermodynamic grounds. A detailed revisit of the EEC phenomenon on varied kinetic and equilibrium processes has been also presented. Possible correlations among the free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes of different similar and nonsimilar chemical processes under varied conditions have been discussed with possible future projections.

Porin differentiates TLR mediated proinflammatory response of follicular zone B cell from TLR-unresponsive IL-10 expressing marginal zone B cell.

TLR-ligands are frequently chosen as candidates for vaccine or adjuvant development because they can primarily bridge innate signaling with adaptive immune responses. Since the adjuvant action of porin, the major outer membrane protein commonly present on Gram-negative bacteria, has been tested on several antigen-presenting cells, we investigated its role in driving systemic immunity which is considered a benchmark for a successful adjuvant. Here, we show porin differentially regulated splenic marginal zone (MZ) and follicular zone (FO) B cell responses in contrast to other classical TLR2-ligands FSL-1 and Pam3CSK4. The protein up-regulated TLR2 and TLR6 and stimulated the activation and costimulatory molecules on FO B cells skewing the cells toward TLR-dependent type-1 cytokine response. However, porin could not up-regulate the TLRs and activate MZ B cells. These cells responded to porin by expressing toll-interacting protein (TOLLIP), the TLR2 and -4 signaling inhibitor along with stimulation of the intracellular pathogen recognition receptor NLR caspase recruitment domain containing protein 5 (NLRC5). The CD1d(hi) MZ B cells released IL-10 unequivocally demonstrating regulatory B cell feature. Immunization with porin also resulted in transient IL-10 expression by the CD19(+)CD21(hi) B cells prior to plasma cell formation. Moreover, the plasma cells developed from the B-2 cell subsets show marked variation in generation of immunoglobulin subclasses. The work delineates multi-faceted role of B cell subsets induced by porin for robust immunity without compromising with the checks and controls.

LPS stimulates and Hsp70 down-regulates TLR4 to orchestrate differential cytokine response of culture-differentiated innate memory CD8(+) T cells.

Nonconventional innate memory CD8(+) T cells characteristically expressing CD44, CD122, eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) were derived in culture from CD4(+)CD8(+) double positive (DP) thymocytes of normal BALB/c and C57BL/6 mice. These culture-differentiated cells constitutively express toll-like receptor (TLR)4 and release interferon (IFN)-γ and interleukin (IL)-10. We show the TLR4-ligand lipopolysaccharide (LPS) stimulate the TLR and up-regulate IFN-γ skewing the cells towards type 1 polarization. In presence of LPS these cells also express suppressor of cytokine signaling (SOCS)1 and thus suppress IL-10 expression. In contrast, heat shock protein (Hsp)70 down-regulated TLR4 augmenting the anti-inflammatory cytokine IL-10. In association with IL-10 release IFN-γ was abrogated. The programmed cell death (PD)-1 mostly present in regulatory T cells was stimulated in these IL-10 producing cells by Hsp70 and not LPS indicating the cells can be driven to two contrast outcomes by the two TLR4 ligands. Our work provides a scope for in vitro monitoring of CD8(+) T cells to decipher important immune therapeutic option during infection or sepsis.

Activation of TOLLIP by porin prevents TLR2-associated IFN-γ and TNF-α-induced apoptosis of intestinal epithelial cells.

Interferon (IFN)-γ and tumor necrosis factor (TNF)-α cause chronic inflammation of the intestine leading to progression of inflammatory bowel disease (IBD), which is manifested through rapid apoptosis of the intestinal epithelial cells (iECs). Here, we show inhibition of IFN-γ and TNF-α-induced apoptosis of INT-407 cells by porin, a microbe-associated molecular pattern (MAMP) with affinity for toll-like receptor (TLR)2 and commonly present in Gram-negative bacteria. Proinflammatory cytokines induce apoptosis by activation of caspase 8 that triggers caspase 9 through Bax finally leading to activation of caspase 3, the executioner caspase. Interestingly, while IFN-γ and TNF-α promotes Bax expression, in contrast porin up-regulates anti-apoptotic Bcl-xL resulting in iEC survivability. We show elevated expression of TLR2 is a key requisite for IFN-γ and TNF-α mediated caspase 8 up-regulation that contributes to apoptosis of iECs. Down-regulation of TLR2 expression is central for checking apoptosis which is achieved by elevated level of toll-interacting protein (TOLLIP) in presence of porin. Attempts to limit IBD is in progress with anti-IFN-γ and anti-TNF-α Abs or use of IL-10. Although probiotic bacterial proteins have shown to successfully reduce IFN-γ and TNF-α mediated apoptosis, the exact mechanism of their action has remained elusive. This study identifies the underlying sequential events of transient TLR2 stimulation followed by its blocking in response to the bacterial outer membrane protein, which advocates intervention at TLR-juncture is crucial for controlling IBD.

Antigenic relatedness defines Toll-like receptor 2 is crafted on ligand blueprint.

Toll-like receptors are located particularly on mammalian immune cells to recognize pathogen-associated molecules. Toll-like receptors are categorized on the basis of ligand specificity that includes Toll-like receptor 2 with affinity for bacterial porin, the major outer membrane protein. Here we show TLR2 antibody recognizes the monomer of porin, primarily a TLR2-ligand in Western blot, thus displaying relatedness of primary structures between the receptor and its ligand. Quantitative analysis revealed relatedness of the native porin molecule with TLR2 was as high as 71%, suggesting imprint of native porin trimer is mostly copied by the receptor crossing limits of primary structures. Flow cytometric analysis of TLR2 on HEK-293 cells shows the receptor and ligand also have common molecular patterns on surface, which is distinctively separate from regions assigned for putative TLR(*)ligand interaction. Molecular mimetic and specificity of TLR will caution investigators targeting TLR-ligands to develop adjuvants and vaccines.

Bacterial ligand stimulates TLR2-dependent chemokines of colon cell.

Shigella spp. are known to penetrate the colonic epithelial cells causing shigellosis, which results in production of convalescent antibodies against porin, the surface exposed major outer membrane protein. Porin has been categorized as primarily TLR2-ligand and here we validated its signaling procedure in colonic INT-407 cells simulating the host scenario. Porin up-regulated TLR2 and -6 followed by TLR2·MYD88 complex formation suggesting direct involvement of MYD88 for downstream signaling. Translocation of NF-κB p65 and p50 subunits on to the nucleus indicates involvement of the transcription factor in signaling. Porin-induced TLR signaling specifically stimulated the pro-inflammatory chemokine panel comprising of MIP-1α, MCP-1 and IL-8. Inhibition studies of TLR2 and NF-κB led to abrogation of the pro-inflammatory chemokine response, showing TLR-dependent signaling through NF-κB regulate gut activity. This work elucidates TLR2 not only scans pathogen-associated molecule but also has a direct role in maneuvering colon cell response.

Culture-differentiated CD8(+) T cells acquire innate memory-like traits and respond to a pathogen-associated molecule.

Selection of conventional CD4(+) or CD8(+) T cells is usually driven by the interaction of double-positive CD4(+)CD8(+) thymocytes with epithelial cells. Here, we demonstrate preferential selection of CD8(+) thymocytes from in vitro differentiation of CD4(+)CD8(+) double-positive thymocytes exhibiting the characteristics of nonconventional innate memory CD8(+) cells. In contrast to conventional CD8(+) thymocytes, these culture-differentiated CD8(+) cells are eomesodermin positive and robustly express CXCR3, CD44, CD122 and TLR2. Interestingly, the pathogen-associated molecule porin promotes preferential differentiation of the CD8(+) single-positive subset in association with promyelocytic leukemia zinc-finger upregulation and interleukin (IL)-4 production. On priming with anti-CD3 antibody, porin augmented TLR2 and IFN-γ indicating a role of the TLR ligand in acquisition of innate memory response of CD8(+) thymocytes. In addition, porin has a cooperative role with IL-15 on the expansion of memory-phenotype CD8(+) T cells along with its effector function. Thus, the study opens an avenue to unfold the cues for development of these cells and the strategies adopted for bolstering immunity during primary infection.

Alternative TLRs are stimulated by bacterial ligand to induce TLR2-unresponsive colon cell response.

Although pathogenic bacteria penetrate colonic cells causing infection, the role of its surface molecules serving as key Toll-like receptor (TLR) ligands and triggering response remains unexplored. We show that TLR2-ligand porin up-regulated TLR4 on HT-29 cells, which the TLR4-ligand LPS could not. TLR1 that co-express with TLR2 got stimulated with TLR4. Besides the two TLRs, MD-2 was expressed revealing that the TLR4 co-receptor is not exclusive for LPS signaling. SARM-1 that mostly down-regulates TLR-signaling, demonstrated central role in signaling by engaging IRF-3 and NF-κB for cell activity. Porin induced type 1 chemokines particularly MCP-3, while porin-stimulated HT-29 culture supernatant displayed PBMC migration, collectively suggesting that the chemokines influence colon and immune cell cross-talk. In TLR2 down-regulated HT-29 cells, we found TLR1 and TLR4 as substitute TLRs to identify porin and orchestrate signaling. Thus, TLR replacement for PAMP recognition demonstrates specificity of ligand·TLR association can compromise and is a necessary alternative for successful execution of immune responses.

Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages.

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the ß-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

IL-10 alters prolactin receptor activity emulating that during breast cancer.

Peripheral blood mononuclear cells (PBMC) of breast cancer patients show altered prolactin (PRL)-induced proinflammatory response and express short form of prolactin receptor (PRL-R), besides secreting elevated level of interleukin (IL)-10 than that of the normal counterparts. IL-10 depleted the functional long form of PRL-R mRNA and protein, expressed PRL-R (SF) mRNA and blocked the PRL response found in normal individuals, which could be a mechanism to suppress the proinflammatory immune responses during malignancy.

Exploring the potential of Nelumbo nucifera rhizome on membrane stabilization, mast cell protection, nitric oxide synthesis, and expression of costimulatory molecules.

Immunomodulatory activity of Nelumbo nucifera rhizome was evaluated for its standardized extract (NNRE) with respect to betulinic acid. Various key parameters including erythrocyte membrane stabilization, inhibition of histamine release, reduction in nitric oxide production and depletion of expression of costimulatory molecules of macrophages were estimated. The result displayed that NNRE stabilized erythrocyte membrane significantly at 10 (42.05%) and 100 microg/mL (44.31%). Although considering the protection of mast cells from degranulation, NNRE showed 38.66% (100 microg/mL) and 69.66% (10 microg/mL) degranulation against compound 48/80 (C 48/80). NNRE at 1 and 5 microg/mL inhibited lipopolysaccharide (LPS)-induced activation of macrophages by decreasing the expression of costimulatory molecules. Expression of CD40, CD80, and CD86 by NNRE was seen significantly at 5 microg/mL compared to LPS-treated group. The extracts also inhibited the nitrite concentration at 1 and 5 microg/mL compared to LPS-treated group.

Porin of Shigella dysenteriae directly promotes toll-like receptor 2-mediated CD4+ T cell survival and effector function.

Porin of Shigella dysenteriae type 1 up-regulated Toll-like receptor (TLR)2 on CD3-stimulated CD4(+) T cells but could not induce the expression of other TLRs. TLR2 in association with myeloid differentiating factor 88 (MyD88) triggered the downstream signal transduction pathway leading to activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB), and degradation of IkappaB, the NF-kappaB inhibitor. TLR2 co-stimulation by porin resulted in T cell expansion by inducing both proliferation and survival of the CD4(+) T cells. Extracellular signal-regulated kinase (ERK)1/2 activation inhibitor U0126 and NF-kappaB translocation inhibitor SN-50 significantly inhibited proliferation of T cells, highlighting a direct role of ERK and NF-kappaB in the process. However, cell survival involving Bcl-X(L) induction was found to be regulated essentially by ERK with no significant role of NF-kappaB. Porin-induced proliferation was supported by induction of IL-2 and CD25 that are known to play a pivotal role in T cell expansion. Apart from inducing T cell proliferation, porin triggered effector functions of the cells, evident from TLR2- and MyD88-dependent release of type 1 cytokines tumor necrosis factor (TNF) and interferon (IFN)-gamma along with the induction of type 1 chemokines macrophage-inflammatory protein (MIP)-1alpha and MIP-1beta and their receptor CCR5. The proliferation, survival and effector function of CD4(+) T cells through TLR2 co-stimulation show the capability of porin to directly turn adaptive immunity into action.