Dietary Application of the Microalga Lobosphaera incisa P127 Reduces Severity of Intestinal Inflammation, Modulates Gut-Associated Gene Expression, and Microbiome in the Zebrafish Model of IBD.

SCOPE: Microalgae are an emerging nutritional resource of biomolecules with potential to alleviate gut inflammation. The study explores the anti-inflammatory and immunomodulatory potential of the microalga Lobosphaera incisa P127, which accumulates a rare omega-6 LC-PUFA dihomo-É£-linolenic acid (DGLA) under nitrogen starvation. The therapeutic potential of dietary supplementation with P127 is investigated in the zebrafish model of IBD (TNBS-induced colitis). METHODS AND RESULTS: Guts are sampled from zebrafish fed experimental diets for 4 weeks, before and 24 h after TNBS challenge. Diets containing 15% non-starved (Ns) and 7.5% and 15% N-starved (St) algal biomass significantly attenuate the severity of gut injury and goblet cell depletion. In contrast, diets containing 7.5% Ns and DGLA ethyl ester have no effect on gut condition. Fish fed 15% St, high-DGLA biomass, have the fewest individuals with pathological alterations in the gut. Dietary inclusion of Ns and St distinctly modulates gut-associated expression of the immune and inflammatory genes. Fish fed 15% Ns biomass display a coordinated boost in immune gene expression and show major changes in the gut microbiome prior challenge. CONCLUSION: Dietary inclusion of L. incisa biomass at two physiological states, ameliorates TNBS-induced gut inflammation, suggesting the synergistic beneficial effects of biomass components not limited to DGLA.

Simulation of high-Mach-number inviscid flows using a third-order Runge-Kutta and fifth-order WENO-based finite-difference lattice Boltzmann method.

A discrete-velocity Boltzmann equation (DVBE) with Bhatnagar-Gross-Krook (BGK) approximation is discretized in time and space using a third-order Runge-Kutta (RK3) and fifth-order weighted essentially nonoscillatory (WENO) finite-difference scheme to simulate benchmark inviscid compressible flows. The implementation of the WENO ensures that solutions behave with minimal or no oscillations, narrowing the gap between the exact and the numerical results. Discrete-velocity sets given by Kataoka and Tsutahara [Phys. Rev. E 69, 056702 (2004)10.1103/PhysRevE.69.056702] are used. The additional dissipation terms as well as artificial viscosity are incorporated in the formulation to solve the compressible flows at high Mach number. Further, the flows which are subjected initially to a high density ratio are effectively simulated. In this article, one-dimensional benchmarks are simulated at initial Mach number up to 30 and density ratio up to 1000, whereas, the benchmarks in two dimensions are simulated with a Mach number up to 10. The algorithm is assessed by simulating numerous benchmarks, namely, (i) one-dimensional Riemann problem for various shock waves combinations [namely (a) shock-shock waves in the case of different Mach numbers, (b) rarefaction-shock waves for various density ratios, (c) sudden contact shock discontinuity, and (d) shock-rarefaction waves for density ratio 5], (ii) isentropic vortex convection test, (iii) regular shock reflection (RR) for Mach numbers 2.9 and 10, (iv) double Mach reflection (DMR) for inflow Mach numbers as 6 and 10, and (v) forward-facing step for inflow Mach numbers 2 to 5. Further, the effect of change in Mach numbers and wedge angles on the flow structures in the case of DMR are detailed. In the case of a forward-facing step, the variations of flow structure (e.g., the Mach stem height, triple points location, and shock standoff distance) are detailed with respect to Mach number, step height, and specific-heat ratios. Finally, the numerical stability of the proposed formulation is carried out to assess the behavior of the free parameters.

Identification of miRNA and their target genes in Cestrum nocturnum L. and Cestrum diurnum L. in stress responses.

MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules and products of primary miRNAs that regulate the target gene expression. Homology-based approaches were employed to identify miRNAs and their targets in Cestrum nocturnum L. and Cestrum diurnum L. A total of 32 and 12 miRNA candidates were identified in C. nocturnum and C. diurnum. These miRNAs belong to 26 and 10 miRNA families and regulate 1024 and 1007 target genes in C. nocturnum, and C. diurnum, respectively. The functional roles of these miRNAs have not been earlier elucidated in Cestrum. MiR815a, miR849, miR1089 and miR172 have a strong propensity to target genes controlling phytochrome-interacting factor 1 (PIF1), ubiquitin-specific protease 12 (UBP12), leucine-rich repeat (LRR) protein kinase and GAI, RGA, SCR (GRAS) family transcription factor in C. nocturnum. While miR5205a, miR1436 and miR530 regulate PATATIN-like protein 6 (PLP6), PHD finger transcription factor and myb domain protein 48 (MYB48) in C. diurnum. Overall, these miRNAs have regulatory responses in biotic and abiotic stresses in both plant species. Eight putative miRNAs and their target genes were selected for qRT-PCR validation. The validated results suggested the importance of miR815a, miR849, miR5205a, miR1089, miR172, miR1436, and miR530 in exerting control over stress responses in C. nocturnum and C. diurnum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01127-1.

High presacral collection approached through the perineal route: A novel computed tomography-guided technique.

For a definitive diagnosis of abdomino-pelvic lesions, percutaneous aspiration or biopsy is often necessary; however, finding a safe 'window' for access is challenging. This case report discusses a novel method to approach a deep pelvic collection and also briefly reviews the various approaches to access such lesions. A sample was obtained from a non-resolving presacral collection using a CT-guided percutaneous, trans-perineal approach with repeated sessions of hydro-dissection. Successful aspiration and analysis revealed multi-drug resistant tuberculosis, thus guiding appropriate management.

Dietary Supplementation with Omega-6 LC-PUFA-Rich Microalgae Regulates Mucosal Immune Response and Promotes Microbial Diversity in the Zebrafish Gut.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism - cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine - interleukin 10 (il-10), immune defense - lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants - catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1%-1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

The gut mucosal barrier of zebrafish (Danio rerio) responds to the time-restricted delivery of Lobosphaera incisa-enriched diets.

Recent studies in mammalian models revealed compelling evidence that along with the intrinsic characteristics of diets, the time of their delivery could have a profound impact on their benefits. In this study, we explored a time-dependent modulation of the gut mucosal barrier by delivering diets enriched with the green microalga (Lobosphaera incisa) either in a time-restricted regime or randomly to zebrafish (Danio rerio). The basal diet was enriched with microalgal biomass through two inclusion levels (i.e., 10% and 15% w/w), and the feeding trial lasted for six weeks. The control group was fed with the basal diet. After collection of tissue samples at week 6, the remaining fish were challenged by intraperitoneal injection of Streptococcus inaie. A histological analysis of the gut structure revealed that the fish that received the microalgae randomly exhibited shorter villi length. Genes coding for immunity were modulated in the gut by dietary treatments. Notably, the transcript levels of lysozyme, ß-defensin and hepcidin were significantly higher in the group subjected to the time-restricted feeding regime. Dietary microalgae affected the fatty acid content in the gut, particularly the level of arachidonic acid (ARA), and the time-restricted feeding influenced its accumulation. Groups that received diets enriched with 15% microalgae, regardless of the feeding strategy, displayed a significantly higher resistance to S. inaie 16 days post-infection, though differences between the delivery strategies were pronounced during the early stage of infection. In conclusion, the dietary inclusion of L. incisa modulated some of the features of the gut mucosal barrier of zebrafish, and the time of delivery appeared to have a considerable influence on immunomodulatory functions.

Dietary Supplementation With ω6 LC-PUFA-Rich Algae Modulates Zebrafish Immune Function and Improves Resistance to Streptococcal Infection.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells.

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.

Dietary arachidonic acid affects immune function and fatty acid composition in cultured rabbitfish Siganus rivulatus.

The marbled spinefoot rabbitfish (Siganus rivulatus) is an economically valuable fish species that has potential for commercial production in aquaculture. To overcome challenges in its sustainable production, a formulated diet is required for imparting health and robustness. This study evaluates the effect of dietary supplementation with arachidonic acid (ARA; 20:4n-6) on growth, survival, immune function and fatty acid composition of red blood cells (RBCs) in rabbitfish. We conducted two feeding trials using juvenile fish (to evaluate growth and survival) and adults (to evaluate immune function and fatty acid incorporation). Fish were fed diets supplemented with three different levels of ARA (in % of total fatty acids): 0.6 (unsupplemented control), 2.6 (moderate) and 4.7 (high). The fish fed with moderate ARA levels exhibited improved (p < 0.05) growth over the control and the high ARA level groups. During an outbreak of Streptococcus iniae, fish fed with moderate ARA survived significantly (p < 0.05) better (89%) than the control and the high ARA groups (59% and 48%, respectively). Moderate ARA supplementation resulted in elevated lysozyme and complement levels in the plasma of rabbitfish. A significant increase in the total serum immunoglobulin levels was observed in both the medium and the high ARA level groups; however, a decrease in antiprotease activity was recorded in the supplemented groups as compared to the control. Fatty acid analysis in fish red blood cells revealed a significant (p < 0.05) increase in the proportion of ARA of total fatty acids in the groups fed with the medium and the high ARA level diets (9.5% and 11.2%, respectively, compared to 7.1% in the control). Concomitantly, there was a decrease in the proportion of eicosapentaenoic acid (EPA; 20:5n-3), dihomo-γ linolenic acid (DGLA; 20:3n-6) and several 18-carbon unsaturated fatty acids in these groups. In conclusion, ARA in rabbitfish feeds improved growth, survival as well as innate and acquired humoral immune functions. Thus ARA supplementation in the diet of this species could be a valuable step towards establishing the commercial culture of rabbitfish.

PCR detection of ansA from marine bacteria and its sequence characteristics from Bacillus tequilensis NIOS4.

As many as 71 marine bacterial DNA extracts were PCR screened for L-asparaginase (ansA), a key gene in anti-cancer molecular-searches. Over 62% (44) of them were positive for ansA gene. The positive cultures were from genera Bacillus and Staphylococcus. The ansA gene cloned from isolate NIOS4 belonging to recently described Bacillus tequilensis is 1099 bp in length with a 990 bp ORF coding for 329 amino acids. BLASTx analysis revealed this sequence to be 98% similar to earlier reported ansA sequence from B. subtilis (Accession no. NP390239.1). By comparing its deduced amino acid sequence with other bacterial asparaginase sequences six substitutions at positions 305(Thr), 313(Lys), 314(Leu), 315(Asp), 318(Arg), and 320(Gln) are observed. Key residues like Thr(12), Thr(85), Asp(86), Lys(156), and Phe(165) taking part in active-site formation and imparting catalytic properties are conserved. The phylogenetic tree based of the ansA amino acid sequences revealed close relatedness of the NIOS4 ansA sequence with B. subtilis (Accession no. NP 390239.1). It's very close genetic resemblance to B. subtilis and conservation of certain key amino acid residues suggest it as a prospective candidate for evaluation and, production of L-asparaginases.