Immunity to severe malaria is acquired quickly, operates independently of pathogen load, and represents a highly effective form of disease tolerance. The mechanism that underpins tolerance remains unknown. We used a human rechallenge model of falciparum malaria in which healthy adult volunteers were infected three times over a 12 mo period to track the development of disease tolerance in real-time. We found that parasitemia triggered a hardwired innate immune response that led to systemic inflammation, pyrexia, and hallmark symptoms of clinical malaria across the first three infections of life. In contrast, a single infection was sufficient to reprogram T cell activation and reduce the number and diversity of effector cells upon rechallenge. Crucially, this did not silence stem-like memory cells but instead prevented the generation of cytotoxic effectors associated with autoinflammatory disease. Tolerized hosts were thus able to prevent collateral tissue damage in the absence of antiparasite immunity.

Diabetic nephropathy (DN) is one of the leading causes of end-stage renal disease. The most popular biomarkers in current research on DN are microRNAs. There are studies showing that while the expression of SOX6 increases, hsa-miR-342-3p expression decreases in kidney tissues with DN. The current study evaluated hsa-miR-342-3p expression by Real Time PCR and SOX6 expression by immunohistochemistry in a cohort of 110 DN biopsies, as well as their relationship with various clinical and histomorphological parameters. An inverse relationship between expression of hsa-miR-342-3p and SOX6 was demonstrated. SOX6 genetic expression was correlated with serum creatinine and tubular basement membrane thickening. Immunohistochemically, SOX6 staining was observed in mesangial cells and podocytes in 21 patients, with tubular staining in 45, and interstitial staining in 27 patients. Tubular staining was associated with proteinuria, interstitial fibrosis and inflammation; interstitial staining was associated with creatinine; and staining in the glomerular compartment was associated with advanced DN class. Our study is the first in the literature in which SOX6 was applied immunohistochemically in human kidney tissue, and its relation with DN classes was examined. We demonstrate its correlation with laboratory and histomorphological parameters, and provide a rational basis for future studies on larger patient groups that may result in the development of new biomarkers to predict the progression of DN and enhance its treatment.

The regulation of the cyclic oscillation of the components of the circadian clock is complex in itself. Numerous clock interactions with processes and molecules present in cells further complicate this mechanism. Recently, the anti-aging protein Silencing Information Regulator Two family member, SIRT1, has been linked with the molecular circadian clock. In this study, we investigated the in vitro effect of aminooxyacetic acid on SIRT1 expression in relation to circadian dynamics of Cry1 and Bma11 expressions in serum shocked NIH-3 T3 and HaCaT cells. The study was carried out in the context of the inhibitory activity of aminooxyacetic acid against cystathionine-β-synthase and cystathionine-γ-lyase. We have shown that aminooxyacetic acid effectively inhibits SIRT1 transcription and synthesis, which, given the pleiotropic effects of sirtuin 1 on numerous metabolic pathways, may have other implications. We also found that AOAA contributes to up-regulation of the expression of the Cry1 and Bmal1 genes in cells. This effect does not appear to be related to inhibition of the activity of cystathionine-β-synthase and cystathionine-γ-lyase. At the same time, this does not deny the role of hydrogen sulphide, a product of the activity of these enzymes, in the regulation of the circadian clock.

Modified tRNA anticodons are critical for proper mRNA translation during protein synthesis. It is generally thought that almost all bacterial tRNAsIle use a modified cytidine-lysidine (L)-at the first position (34) of the anticodon to decipher the AUA codon as isoleucine (Ile). Here we report that tRNAsIle from plant organelles and a subset of bacteria contain a new cytidine derivative, designated 2-aminovaleramididine (ava2C). Like L34, ava2C34 governs both Ile-charging ability and AUA decoding. Cryo-electron microscopy structural analyses revealed molecular details of codon recognition by ava2C34 with a specific interaction between its terminal amide group and an mRNA residue 3'-adjacent to the AUA codon. These findings reveal the evolutionary variation of an essential tRNA modification and demonstrate the molecular basis of AUA decoding mediated by a unique tRNA modification.

Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

Biofortified maize with enhanced carotenoid content was developed to combat vitamin A deficiency. However, it was observed that during storage, carotenoids present in maize grain get degraded and it has been reported that carotenoid cleavage dioxygenase1 (ccd1) is responsible for this degradation.

In our current study, comprehensive in-silico analysis deciphered a complete overview of the ccd1 gene in maize including the gene structures, phylogeny, chromosomal locations, promoter analysis, conserved motifs and interacting protein partners. In addition to these, a comparative in-silico analysis of the ccd1 gene in maize, rice and Arabidopsis was performed. An intronic region of ccd1, unique to the maize genome, was matched significantly with a lot of long non-coding RNA and was identified. Also, growth stage-specific ccd1 expression analysis was performed in two maize inbred lines, V335PV and HKI161PV. The results indicate that both inbred lines displayed higher ccd1 expression during reproductive stages compared to vegetative stages, with the highest expression level observed at the milking stage in both inbreds.

This detailed in-silico characterisation and expression analysis of the ccd1 gene contributes to our understanding of its activity and expression pattern in maize in stage and tissue-specific manner. This study will further provide an effective strategy for manipulating the ccd1 gene to enhance the carotenoid content of maize grain, thereby aiding in the combat against vitamin A deficiency.

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GC's spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GC's spatial heterogeneity. SURF6's association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates-suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

Macroautophagy/autophagy is crucial for cell survival during nutrient starvation. Autophagy requires the coordinated function of several Atg proteins, including the Atg1 kinase, for efficient induction and execution. Recently, several RNA-binding proteins (RBPs) have been shown to post-transcriptionally regulate ATG1. However, a comprehensive understanding of autophagy regulation by RBPs via ATG1 is yet to be elucidated. Here, we utilize an in vitro approach to identify RBPs that specifically interact with ATG1 untranslated regions. We show that Npl3 and Pub1 interact with the ATG1 5' and 3' untranslated regions during nitrogen starvation. Furthermore, Npl3 and Pub1 coordinate to facilitate ATG1 mRNA export to the cytoplasm and its subsequent interaction with the translational machinery. Significantly, in non-small cell lung cancer cell lines, mammalian Pub1, TIA1, also positively regulates ULK1 protein expression and autophagy during serum starvation. Overall, our study highlights the regulatory landscape that fine-tunes Atg1 protein expression to sustain autophagy during nutrient starvation.

Human studies suggest that heavy alcohol use may be an etiological factor contributing to the development of Alzheimer's disease (AD) neuropathology. Both alcohol use disorder (AUD) and AD share common underlying neuropathology, including proinflammatory high-mobility group box 1 (HMGB1)-mediated neuroimmune signaling and basal forebrain cholinergic neuron degeneration. Adolescent onset of binge drinking represents a significant risk factor for later development of an AUD, and accumulating evidence suggests that adolescent initiation of heavy alcohol use induces HMGB1 signaling and causes degeneration of the basal forebrain cholinergic system that persists into adulthood. However, it is unknown whether adolescent binge drinking confers increased risk for later development of AD-associated neuropathology through persistent induction of proinflammatory HMGB1 neuroimmune signaling. To investigate this question, we first (Experiment 1) assessed AD-associated neuropathology in the post-mortem human basal forebrain of individuals with AUD and an adolescent age of drinking onset relative to age-matched moderate drinking controls (CONs). In Experiment 2, we treated non-transgenic and 5xFAD male and female mice, which overexpress both mutant human APP and PS1, with adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g. 2-days on/2-days off; postnatal day [P]30 - P55), and assessed AD-associated neuropathology in the adult (P100) basal forebrain. In Experiment 3, 5xFAD female mice received AIE treatment followed by glycyrrhizic acid (150 mg/L), an HMGB1 inhibitor, in drinking water from P56 to P100, and basal forebrain tissue was collected on P100 for assessment of AD-associated neuropathology. In the post-mortem human AUD basal forebrain (Experiment 1), we report upregulation of Hmgb1 and the HMGB1 receptors Rage and Tlr4 as well as microglial activation and increased intraneuronal Aβ1-42 accumulation in association with reduced cholinergic neuron marker expression (ChAT). In the 5xFAD mouse model (Experiment 2), AIE accelerated AD-associated induction of Hmgb1 proinflammatory neuroimmune genes, microglial activation, and reductions of ChAT+ basal forebrain cholinergic neurons in the adult female, but not male, basal forebrain. In Experiment 3, post-AIE treatment with glycyrrhizic acid rescued the AIE-induced acceleration of AD-associated increases in proinflammatory HMGB1 neuroimmune signaling, microglial activation, and persistent reductions of basal forebrain cholinergic neurons in adult 5xFAD female mice. Together, these findings suggest that adolescent binge ethanol exposure may represent an underappreciated etiological factor contributing to onset of AD-associated neuropathology in adulthood through HMGB1- mediated neuroimmune signaling.

The search has been ongoing for safe and effective antimicrobial agents for control and prevention of oral biofilm associated with disease. Clinical trials for oral specific anti-bacterials are costly and often provide inconclusive results. The simple approach of ex vivo testing of these agents has not demonstrated utility, likely due to variability of effects observed even with a single donor. We show how shed oral biofilms, easily obtained from donor saliva, and tested under optimized conditions, respond reproducibly to anti-bacterial challenges measured by reductions in rRNA accumulation in susceptible taxa. Responses are in part donor specific, but many bacteria taxa were shown to be reproducibly susceptible over a group of donors. For two antibiotics, vancomycin and penicillin G tested at pharmacologic levels, a subset of Gram-positive bacteria was inhibited. A natural product with antibacterial properties, diluted Vaccinium macrocarpon (cranberry) juice, was shown to inhibit a range of oral taxa, including Alloprevotella sp__HMT_473, Granulicatella adiacens, Lachnoanaerobaculum umeaense, Lepotrichia sp__HMT_215, Peptostreptococcus stomatis, Prevotella nanceiensis, Stomatobaculum sp__HMT_097, Veillonella parvula, and kill some targets. The model discussed in this study has promise as a rapid, precise, and reproducible ex vivo method to test and identify potential clinically useful antimicrobial agents active against the oral biofilm community.

L-Arginine supplementation has beneficial effects on metabolic disorders in rodents. We here investigated the effects of exogenous L-arginine on cardiac pathology and mitochondrial reactive oxygen species (ROS) production and dynamics in DahlS.Z-Leprfa/Leprfa (DS/obese) rats, a model of metabolic syndrome (MetS). DS/obese rats and their lean homozygous littermate (DahlS.Z-Lepr+/Lepr+, or DS/lean) controls were provided with drinking water containing 0.50% L-arginine-HCl or 0.85% L-alanine (isonitrogenous control) from 13 to 17 weeks of age. L-Arginine supplementation markedly alleviated hypertension without affecting cardiac injury in MetS rats. It also attenuated the increase in ROS production apparent in cardiac mitochondria isolated from MetS rats as well as suppressed the associated upregulation of Nox4 mRNA and protein in the heart. Furthermore, L-arginine reversed the decrease in the size of cardiac mitochondria as well as changes in the expression of DRP1 and OPA1 proteins apparent in the L-alanine-treated MetS rat heart. Cardiac arginase II gene expression and arginase activity were increased by L-arginine treatment in MetS rats but not CONT rats. L-Arginine supplementation thus ameliorated hypertension and cardiac mitochondrial abnormalities in MetS rats, with the lack of a cardioprotective effect possibly being due to increased arginase activity.

While mutations in the PIK3CA gene play important roles in human breast carcinogenesis, PIK3R1 gene alterations are recognized as actionable mutations for clinical cancer treatment. We aimed to elucidate the role of PIK3R1 in cell proliferation on breast carcinoma and to correlate the PIK3R1 expression with patients' outcome using human tumor tissue arrays.

Using human BT-474 (estrogen receptor (ER)+/human epidermal growth factor receptor 2 (HER2)-high) breast carcinoma cell line as in vitro model, the role of PIK3R1 in cell proliferation was elucidated by knock-down of the PIK3R1 gene (ΔPIK3R1) in this cell line. Between January 2000 to December 2015, the records of a cohort of 440 patients in our hospital were retrospectively reviewed, including patients' survival. The correlations between PIK3R1 expression and patient prognosis, such as overall survival (OS) and disease-free survival (DFS), were elucidated by human breast cancer tumor tissue array immunostaining.

After the PIK3R1 gene was silenced in the BT-474 line, there was an increased cell number and a decrease in the G0G1-fraction, and increased S-fraction and the S+G2M-fraction for the ΔPIK3R1-BT-474 cell line, as compared to their cell wild type (WT) line. Western blot analysis showed that decreased PIK3R1 protein levels were accompanied by an increase of the p-AKT and p-mTOR proteins in the ΔPIK3R1-BT-474 cell line, compared to the equivalent WT line. Using a human tumor tissue array, patients with high-expressed PIK3R1 protein had better outcomes in terms of DFS and OS, compared to those with low-expressed PIK3R1 protein, when breast cancer was at an early stage (stage I/II), but not across all stages of breast cancer in human patients.

We concluded that downregulated PIK3R1 in BT-474 cells resulted in an increased cell growth and upregulated AKT-mTOR signaling. Clinically, the high-expressed PIK3R1 protein in tumors correlates positively with patients' outcome in stage I and II breast cancer.

Bread wheat serves as an important staple crop in the human diet, largely because of the physicochemical properties of its dough and its protein content. Gluten is the main and complex component of wheat proteins. Despite the significant importance in breadmaking properties, wheat gluten contains some immunogenic peptides capable of triggering a T cell reaction in celiac disease (CD) patients, leading to inflammation in the small intestine. Among gluten proteins, α-gliadins are the most immunogenic components because they possess the primary T-cell stimulating epitopes (DQ2.5-Glia-α1, DQ2.5-Glia-α2, and DQ2.5-Glia-α3), which are primarily located on the D subgenome. Developing new wheat varieties by integrating the D subgenome from various sources is not only useful for introducing low immunogenic gluten, but it can also circumvent the challenging policies arising from the manipulation of wheat genome through transgenic approaches. Here, we performed RNA amplicon sequencing of the most toxic region of alpha-gliadins to analyze the content and composition of CD-related alpha-gliadin epitopes across eight synthetic wheat lines developed from crosses between durum wheat and different Aegilops species containing the D-genome (Ae. tauschii, Ae. crassa, and Ae. ventricosa). By searching the previously identified 121 epitopes and those with one mismatch in our amplicons, we found 54 different α-gliadins epitopes across our genotypes, four of which were new variants. The canonical epitopes were present in all lines, although their expression patterns varied. The occurrence of DQ2.5-Glia-α1a and DQ2.5-Glia-α3 was higher than that of DQ2.5-Glia-α2 and DQ2.5-Glia-α1b across all genotypes. Since a higher quantity of toxic alpha-gliadin epitopes is associated with increased immunogenicity in individuals susceptible to celiac disease, we measured the frequency of the most toxic alpha-gliadin epitopes among different synthetic lines to estimate the overall immunogenic load of our lines. Generally, the immunogenic load of lines with the D-genome originating from Ae. crassa was much lower than those with the D-genome from Ae. tauschii. In this way, the Ae. tauschii derived lines 5 and 6 contained higher levels of toxic alpha-gliadin epitopes, while lines 3, 4, and 7 (derived from Ae. crassa) contained the lowest levels of toxic peptides. We conclude that replacing the bread wheat D-genome with that of the Ae. crassa may help lower the gluten immunogenicity in the deriving synthetic wheat lines.

Colorectal carcinomas (CRCs) are seldom eradicated by cytotoxic chemotherapy. Cancer cells with stem-like functional properties, often referred to as "cancer stem cells" (CSCs), display preferential resistance to several anti-tumor agents used in cancer chemotherapy, but the molecular mechanisms underpinning their selective survival remain only partially understood.

In this study, we used Transcription Factor Target Genes (TFTG) enrichment analysis to identify transcriptional regulators (activators or repressors) that undergo preferential activation by chemotherapy in CRC cells with a "bottom-of-the-crypt" phenotype (EPCAM+/CD44+/CD166+; CSC-enriched) as compared to CRC cells with a "top-of-the-crypt" phenotype (EPCAM+/CD44neg/CD166neg; CSC-depleted). The two cell populations were purified in parallel by fluorescence-activated cell sorting (FACS) from a patient-derived xenograft (PDX) line representative of a moderately differentiated human CRC, following in vivo chemotherapy with irinotecan (CPT-11). The transcriptional regulators identified as differentially activated were tested for differential expression in normal vs. cancer tissues, and in cell populations enriched in stem/progenitor cell-types as compared to differentiated lineages (goblet cells, enterocytes) in the mouse colon epithelium. Finally, the top candidate was tested for mechanistic contribution to drug-resistance by selective down-regulation using short-hairpin RNAs (shRNAs).

Our analysis identified E2F4 and TFDP1, two core components of the DREAM transcriptional repression complex, as transcriptional modulators preferentially activated by irinotecan in EPCAM+/CD44+/CD166+ as compared to EPCAM+/CD44neg/CD166neg cancer cells. The expression levels of both genes (E2F4, TFDP1) were found up-regulated in CRCs as compared to human normal colon tissues, and in a sub-population of mouse colon epithelial cells enriched in stem/progenitor elements (Epcam+/Cd44+/Cd66alow/Kitneg) as compared to other sub-populations enriched in either goblet cells (Epcam+/Cd44+/Cd66alow/Kit+) or enterocytes (Epcam+/Cd44neg/Cd66ahigh). Most importantly, E2F4 down-regulation using shRNAs dramatically enhanced the sensitivity of human CRCs to in vivo treatment with irinotecan, across three independent PDX models.

Our data identified E2F4 and the DREAM repressor complex as critical regulators of human CRC resistance to irinotecan, and as candidate targets for the development of chemo-sensitizing agents.

Chaling wild rice (Oryza rufipogon Griff.) can survive winter due to its extreme cold tolerance, whereas cultivated rice (Oryza sativa L.) cannot. Here, we found that the expression level of OsCYCBL1 decreased relatively less at low temperatures in Chaling wild rice compared with cultivated rice. Transgenic assays of OsCYCBL1 in Nipponbare (Nip) showed that overexpression of OsCYCBL1 promoted cold tolerance. Transcriptome profiling, RT-qPCR analysis, and physiological parameters measurement indicated that overexpression of OsCYCBL1 maintained better DNA damage repair capacity, balanced the cell cycle, enhanced reactive oxygen species (ROS) homeostasis, and increased wax content, directly affecting the ICE-CBF-COR cascade. Moreover, OsHTR702, a gene that interacts with OsCYCBL1, also positively regulates rice cold tolerance by affecting the ICE-CBF-COR cascade and increasing ROS homeostasis at low temperatures. In addition, overexpression of OsCYCBL1 and OsHTR702 enabled rice to survive through winter. Taken together, the current results indicate that OsCYCBL1 and OsHTR702 are related to cold tolerance in rice, making them potential targets for enhancing crop resilience to cold stress.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

Diversity in the granulocyte repertoire, including neutrophils, basophils, and eosinophils, has been reported in vertebrate species. Medaka fish (Oryzias latipes) have only neutrophils; however, the storage pool of granulopoiesis tissues and the molecular mechanism of granulopoiesis in medaka fish have not been explored. Granulocyte colony-stimulating factor (G-CSF) is a cytokine responsible for neutrophil differentiation, survival, and proliferation. We performed in silico analysis to molecularly characterize the medaka G-CSF and G-CSF receptor (G-CSFR) genes. This study showed that medaka G-CSF differs considerably from human and mouse G-CSF in terms of the primary protein structure; however, the predicted tertiary structure was largely conserved. Analyses of lipopolysaccharide stimulation and G-CSF knockout and overexpression in medaka revealed that G-CSF mobilizes neutrophils into the peripheral blood. The analysis of G-CSF-deficient medaka revealed that G-CSF is involved in erythropoiesis. These findings represent an important first step toward understanding granulocyte hematopoiesis in nonmammalian species.

Background/Objectives: This study explores the potential of using elevated levels of blood double-stranded RNA (dsRNA) as a diagnostic tool for severe fever with thrombocytopenia syndrome (SFTS) infection. Methods: Blood samples from SFTS patients were collected, dsRNA was purified, and total dsRNA expression was quantitatively analyzed using a spiropyran-based method. Comparative analysis was performed using blood samples from healthy individuals and scrub typhus patients with similar symptoms. Results: The results revealed that individuals infected with SFTS had significantly higher total blood dsRNA levels compared to healthy or scrub typhus controls. The dsRNA-based method also has potential for assessing infection severity based on dsRNA levels. Conclusions: These findings suggest that total dsRNA expression can serve as a quick and convenient method to differentiate SFTS from other non-viral conditions with similar clinical presentations. This method shows promise as a novel diagnostic tool.

In recent years a diversity of small noncoding RNAs have been identified that originate from the mitochondrial genome. These mitosRNAs are often dominated by tRNA-derived small RNAs (mito-tDRs). Differential expression of mito-tDRs is associated with responses to stress. They also appear to be expressed differentially during development and their expression may be enriched in stress-tolerant animals. Very little is currently known about roles or modes of action of these sequences, although they are implicated in a diversity of processes such as cell cycle regulation, mRNA stability, regulation of ROS production, and import of proteins into the mitochondrion. To better understand the various roles these sequences may play, it is critical that we understand their diversity, cellular location, and the context for their expression. This protocol outlines the methodologies used to detect mitosRNAs, including mito-tDRs, in embryos and cells of the annual killifish Austrofundulus limnaeus. We highlight critical steps in the isolation of RNA, creation of sequencing libraries, bioinformatics processing of sequence data, and methods for validation of expression that support a robust discovery pipeline for mitosRNAs even from species with incomplete reference genome sequences.

Dectin-1, a C-type lectin, plays important roles in the induction of antifungal immunity. Caspase recruitment domain-containing protein 9 (CARD9) is essential for the dectin-1-induced production of cytokines through the activation of NF-κB. However, the molecular mechanisms underlying the dectin-1-mediated activation of CARD9 have not been fully elucidated. Recently, we reported that the adaptor protein SH3 domain-binding protein 2 (3BP2) is required for the dectin-1-induced production of cytokines and activation of NF-κB, although the relationship between 3BP2 and CARD9 in dectin-1-mediated signaling remains unclear. Here, we report that 3BP2 is required for dectin-1-induced expression of several genes that may contribute to antifungal immunity in bone marrow-derived dendritic cells (BMDCs). The results of reporter assays using HEK-293T cells indicate that 3BP2 induces CARD9-mediated activation of NF-κB through B-cell leukemia/lymphoma 10, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and TNF receptor-associated factor 6-dependent mechanisms. In addition, we show that 3BP2 induces CARD9-mediated ubiquitination of cellular proteins and that MALT1 cleaves 3BP2 in a CARD9-dependent manner. Furthermore, we show that 3BP2 is required for the ubiquitination, in addition to the activation, of MALT1, which leads to MALT1-depenedent cleavage of 3BP2 in dectin-1-stimulated BMDCs. Finally, we identified hematopoietic cell-specific Lyn substrate 1 as a target of 3BP2, which is essential for dectin-1-induced expression of interleukin 10 in BMDCs. These results indicate that 3BP2 regulates gene expression and functions of MALT1 in dectin-1-stimulated cells and that 3BP2 plays an important role in the dectin-1-mediated antifungal immunity.

Ulipristal acetate (UPA) and levonorgestrel are used as emergency hormonal contraceptives. Although both are highly effective in preventing pregnancy, UPA shows efficacy even when taken up to 120 h after unprotected sexual intercourse.

To investigate whether the mechanism of UPA's contraceptive action involves post-fertilization effects.

In vitro and in vivo studies using cultured human endometrial cells and a pre-clinical rat model.

Endometrial cells treated with UPA showed changes in the expression of receptivity gene markers and a significant decrease in trophoblast spheroids attached to the cultured cells. In addition, administration of UPA to female unmated rats decreased the expression of implantation-related genes in the endometrium and inhibited the number of implantation sites in the mated group compared to the non-treated group.

These results support that UPA as an emergency contraceptive might have post-fertilization effects that may affect embryo implantation.

The utilization of human cDNA libraries in yeast genetic screens is an approach that has been used to identify novel gene functions and/or genetic and physical interaction partners through forward genetics using yeast two-hybrid (Y2H) and classical cDNA library screens. Here, we summarize several challenges that have been observed during the implementation of human cDNA library screens in Saccharomyces cerevisiae (budding yeast). Upon the utilization of DNA repair deficient-yeast strains to identify novel genes that rescue the toxic effect of DNA-damage inducing drugs, we have observed a wide range of transcripts that could rescue the strains. However, after several rounds of screening, most of these hits turned out to be false positives, most likely due to spontaneous mutations in the yeast strains that arise as a rescue mechanism due to exposure to toxic DNA damage inducing-drugs. The observed transcripts included mitochondrial hits, non-coding RNAs, truncated cDNAs, and transcription products that resulted from the internal priming of genomic regions. We have also noticed that most cDNA transcripts are not fused with the GAL4 activation domain (GAL4AD), rendering them unsuitable for Y2H screening. Consequently, we utilized Sanger sequencing to screen 282 transcripts obtained from either four different yeast screens or through direct fishing from a human kidney cDNA library. The aim was to gain insights into the different transcription products and to highlight the challenges of cDNA screening approaches in the presence of a significant number of undesired transcription products. In summary, this study describes the challenges encountering human cDNA library screening in yeast as a valuable technique that led to the identification of important molecular mechanisms. The results open research venues to further optimize the process and increase its efficiency.

Aedes aegypti is a major vector of several arboviruses that cause human mortality and morbidity. One method for controlling the spread of these viruses is to control mosquito reproduction. During mating, seminal fluid molecules and sperm are transferred and these stimuli influence female post-mating physiology and behavior. Yet, little is known about the mechanisms underlying these post-mating responses. To fill this gap, short-read RNA sequencing was used to identify differentially expressed genes between unmated (control) and mated females in the head/thorax (HT), abdomen (Ab) and the lower reproductive tract (LRT), of mosquitoes reared with 3% and 12% sucrose. The results revealed that at 3% sucrose, four, 408 and 415 significantly differential expressed genes (DEGs) were identified in the HT, Ab and LRT, respectively, at six hours post mating (hpm). The number of DEGs dropped dramatically at 24 hpm with no DEGs in the HT, three in the Ab, and 112 in the LRT. In contrast, the number of DEGs was lower at 6 hpm than 24 hpm in the LRT at 12% sucrose. Comparing our results to a similar study which used 10% sucrose revealed evidence in support of condition-dependent regulation of gene expression by mating in this species. This study shows that mating-induced transcriptional changes depend on time point after mating, body region, and diet. Our results provide foundational knowledge for future functional analyses to identify genes and pathways involved in the post-mating behavioral and physiological changes of female mosquitoes.

The interaction between the main psychotropic ingredient of Cannabis, Δ⁹- tetrahydrocannabinol (THC), with the endogenous cannabinoid system (ECS) is a critical and underrated issue that deserves utmost attention. The ECS, indeed, contributes to the formation and regulation of excitatory and inhibitory (E/I) neuronal networks that in the hippocampus underly spatial memory. This study explored sex-specific consequences of prenatal exposure to THC in hippocampus-dependent memory and the underlying cellular and molecular contributors of synaptic plasticity and E/I homeostasis. Sprague Dawley dams were exposed to THC (2 mg/kg) or vehicle, from gestational day 5-20. The adolescent progeny of both sexes was tested for: spatial memory retrieval and flexibility in the Barnes Maze; mRNA expression of relevant players of hippocampal synaptic plasticity; density of cholecystokinin-positive basket cells (CCK+BCs) - a major subtype of hippocampal inhibitory interneurons; mRNA expression of the excitatory and inhibitory synaptic proteins neuroligins (Nlgns), as a proxy of synaptic efficiency. Our results show a sex-specific disruption in spatial memory retrieval and flexibility, a male-specific decrease in CCK+BCs density and increase in the expression of markers of neuroplasticity, and consistent changes in the expression of Nlgn-1 and 3 isoforms. Despite a delay in memory retrieval, flexibility of memory was spared in prenatally-THC-exposed female offspring as well as most of the markers of neuroplasticity; a sex-specific increase in CCK+BCs density, and a consistent expression of Nlgn-3 was observed. The current results highlight a major vulnerability to prenatal exposure to THC on memory processing in the male progeny, and sex-specific alterations in the E/I balance and synaptic plasticity.

Biomaterials such as exopolysaccharides have been of great interest for their diverse biological activities in controlling or preventing chronic degenerative diseases, such as cancer. Previously, we isolated four dextrans produced by four strains isolated from Agave salmiana, which were named SF3, SF2, SD1, and SD23. The objective was to evaluate the antitumor activity of these dextrans on prostate (PC3) and colon (SW480) cancer cells. Growth inhibition, morphological changes, mitochondrial metabolism, and cell apoptosis were evaluated by sulforhodamine B, transmission electron microscopy, Seahorse XF and TUNEL assays, respectively. To gene expression was used qPCR and to protein ELISA and immunofluorescence. The cells treated with the dextrans to a dose of 8 mg/mL presented an inhibition of cell growth. Studies of the metabolism cell indicated a disruption in mitochondrial function and a diminished ability of the cells to respond to energy demands through glycolysis. These changes indicate mitochondrial damage resulting in oxidative stress or metabolic alterations. Survivin gen decreased and caspase-3 and -8 increased, key regulators of the apoptotic response with treatment. Moreover, the TUNEL assays indicated cell apoptosis. In conclusion, our findings suggest that dextrans could be considered as potential compounds for cancer treatment.

Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) are economically significant species in Mediterranean countries, serving essential roles in the aquaculture industry due to high market demand and nutritional value. They experience substantial losses from bacterial pathogens Vibrio anguillarum and Streptococcus iniae, particularly at the onset of the summer season. The immune mechanisms involved in fish infections by V. anguillarum and S. iniae remain poorly understood. This study investigated their impact through experiments with control and V. anguillarum- and S. iniae-infected groups for each species. Blood samples were collected at 1, 3, and 7 days post bacterial injection to assess biochemical and immunological parameters, including enzyme activities (AST and ALT), oxidative markers (SOD, GPX, CAT, and MDA), and leukocyte counts. Further analyses included phagocyte activity, lysozyme activity, IgM levels, and complement C3 and C4 levels. Muscle tissues were sampled at 1, 3, and 7 days post injection to assess mRNA expression levels of 18 immune-relevant genes. The focus was on cytokines and immune-related genes, including pro-inflammatory cytokines (TNF-α, TNF-β, IL-2, IL-6, IL-8, IL-12, and IFN-γ), major histocompatibility complex components (MHC-IIα and MHC-IIβ), cytokine receptors (CXCL-10 and CD4-L2), antimicrobial peptides (Pleurocidin and β-defensin), immune regulatory peptides (Thymosin β12, Leap 2, and Lysozyme g), and Galectins (Galectin-8 and Galectin-9). β-actin was used as the housekeeping gene for normalization. Significant species-specific responses were observed in N. Tilapia and E. Sea Bass when infected with V. anguillarum and S. iniae, highlighting differences in biochemical, immune, and gene expression profiles. Notably, in N. Tilapia, AST levels significantly increased by day 7 during S. iniae infection, reaching 45.00 ± 3.00 (p < 0.05), indicating late-stage acute stress or tissue damage. Conversely, E. Sea Bass exhibited a significant rise in ALT levels by day 7 in the S. iniae group, peaking at 33.5 ± 3.20 (p < 0.05), suggesting liver distress or a systemic inflammatory response. On the immunological front, N. Tilapia showed significant increases in respiratory burst activity on day 1 for both pathogens, with values of 0.28 ± 0.03 for V. anguillarum and 0.25 ± 0.02 for S. iniae (p < 0.05), indicating robust initial immune activation. Finally, the gene expression analysis revealed a pronounced peak of TNF-α in E. Sea Bass by day 7 post V. anguillarum infection with a fold change of 6.120, suggesting a strong species-specific pro-inflammatory response strategy. Understanding these responses provides critical insights for enhancing disease management and productivity in aquaculture operations.

Pathogenic fungi employ numerous strategies to colonize plants, infect them, reduce crop yield and quality, and cause significant losses in agricultural production. The increasing use of chemical pesticides has led to various ecological and environmental issues, including the emergence of resistant weeds, soil compaction, and water pollution, all negatively impacting agricultural sustainability. Additionally, the extensive development of synthetic fungicides has adverse effects on animal and human health, prompting the exploration of alternative approaches and green strategies for phytopathogen control. Microorganisms living in sponges represent a promising source of novel bioactive secondary metabolites, potentially useful in developing new nematicidal and antimicrobial agents. This study focuses on extracting bioactive compounds from endosymbiotic bacteria associated with the marine sponge Hyrtios erect sp. (collected from NIOF Station, Hurghada, Red Sea, Egypt) using various organic solvents. Bacillus sp. was isolated and identified through 16 S rRNA gene sequencing. The biocidal activity of Bacillus gotheilii MSB1 extracts was screened against plant pathogenic bacteria, fungi, and nematodes. The n-butanol extract showed significant potential as a biological fungicide against Alternaria alternata and Fusarium oxysporum. Both n-hexane and ethyl acetate extracts exhibited negative impacts against the plant pathogenic bacteria Erwinia carotovora and Ralstonia solanacearum, whereas the n-butanol extract had a positive effect. Regarding nematicidal activity, ethyl acetate and n-butanol extracts demonstrated in-vitro activity against the root-knot nematode Meloidogyne incognita, which causes serious vegetable crop diseases, but the n-hexane extract showed no positive effects. The findings suggest that bioactive compounds from endosymbiotic bacteria associated with marine sponges, particularly B. gotheilii MSB1, hold significant potential as alternative biological control agents against plant pathogens. The n-butanol extract, in particular, displayed promising biocidal activities against various plant pathogenic fungi, bacteria, and nematodes. These results support further exploration and development of such bioactive compounds as sustainable, environmentally friendly alternatives to synthetic pesticides and fungicides in agricultural practices.

Defensins are present in many organisms and are divided into two evolutionary groups, termed cis- and trans-defensins. Cis-defensins have only recently been reported in bacteria, and knowledge of these defensins is limited, with no family classification. Here, we describe the identification of 74 cis-defensins from bacteria and propose five classes for their classification. We also report the first NMR structure determination of a Myxoccocus xanthus defensin, as well as its in silico expression analysis. Xanthusin-1 has a unique structure among the published defensins, which could indicate that the proposed class II peptides constitute a separate group of defensins. Xanthusin-1 gene expression was observed in casitone-based and Streptomyces coelicolor coculture-grown media. Our results demonstrate a wider distribution of defensins outside the Eukarya domain, shedding light on the origin and distribution of defensins. The sharing of three disulfide defensins between bacteria and eukaryotes points to a possible prokaryotic origin of the CSαβ motif. Moreover, the identification of defensins in Gram-positive and Gram-negative bacteria indicates an early origin but with many gene losses during the evolutionary process, similar to findings for eukaryotic defensins.

Septic cardiomyopathy (SCM), a complication initiated by sepsis, presents a significant clinical challenge, leading to increased mortality rates. However, the mechanisms of SCM have not been fully uncovered. Our study involved analyzing RNA sequencing (RNA-seq) data from rat heart tissue, along with utilizing molecular docking and molecular dynamics (MD) simulations, to discover key targets and potential pharmacological actions of the calcitonin gene-related peptide (CGRP) against SCM. A lipopolysaccharide-induced SCM model was established in rats (LPS 10 mg/kg, intraperitoneal (i.p.)). Thereafter, the myocardial tissues from the three groups of rats (Ctrl group, LPS group, and CGRP group) (n = 5) were extracted and underwent RNA-seq, followed by bioinformatics analyses. The qPCR-validated hub targets potentially interacting with CGRP were identified. Following this, homology modeling was utilized to obtain the 3D structure of hub targets, and molecular docking was conducted to evaluate the interaction between CGRP and hub targets. MD simulations (300 ns) were performed to confirm the findings further. Our findings demonstrated that CGRP significantly lowered mortality in SCM rats. 633 DEGs were affected by LPS, contrasted with the Ctrl group. 96 DEGs were affected by CGRP compared to the LPS group. In total, ten fully annotated CGRP-triggered hub genes were obtained. The molecular docking and MD simulations indicate that the relationship between CGRP and eight hub genes is extremely strong. This research offers a thorough examination of the possible objectives and fundamental molecular processes of CGRP in combating SCM, laying the groundwork for investigating the potential protective mechanisms of CGRP against SCM.

Alcohol use disorder (AUD) is defined as patterns of alcohol misuse and affects over 30 million people in the US. AUD is a systemic disease with the epidemiology of acute lung injury and excessive alcohol use established in the literature. However, the distinct mechanisms by which alcohol induces the risk of pulmonary inflammation are less clear. A compelling body of evidence shows that cannabinoid receptors (CB1R and CB2R) play a relevant role in AUD. For this study, we investigated the role of CBR signaling in pulmonary immune activation. Using a human macrophage cell line, we evaluated the expression of CBR1 and CBR2 after cells were exposed to EtOH, +/- cannabinoid agonists and antagonists by flow cytometry. We also evaluated the expression of cannabinoid receptors from the lungs of adolescent mice exposed to acute binge EtOH +/- cannabinoid agonists and antagonists at both resting state and after microbial challenge via western blot, rt-PCR, cytokine analysis, and histology. Our results suggest that EtOH exposure modulates the expression of CBR1 and CBR2. Second, EtOH may contribute to the release of DAMPs and other proinflammatory cytokines, Finally, microbial challenge induces pulmonary inflammation in acute binge EtOH-exposed mice, and this observed immune activation may be CBR-dependent. We have shown that adolescent binge drinking primes the lung to subsequent microbial infection in adulthood and this response can be mitigated with cannabinoid antagonists. These novel findings may provide a framework for developing potential novel therapeutics in AUD research.

Early nutritional challenges can lead to permanent metabolic changes, increasing risk of developing chronic diseases later in life. Total parenteral nutrition (TPN) is a life-saving nutrition regimen, used especially in intrauterine growth-restricted (IUGR) neonates. Early TPN feeding alters metabolism, but whether these alterations are permanent is unclear. Programmed metabolism is likely caused by epigenetic changes due to imbalances of methyl nutrients.

We sought to determine whether feeding TPN in early life would increase risk of developing dyslipidemia in adulthood and whether supplementing the methyl nutrients betaine and creatine to TPN would prevent this development. We also sought to determine whether IUGR exacerbates the effects of neonatal TPN on lipid metabolism in adulthood.

Female piglets (n = 32; 7 d old) were used in 4 treatments: 24 normal-weight piglets were randomly assigned to sow-fed (SowFed), standard TPN (TPN-control), and TPN with betaine and creatine (TPN-B+C); 8 IUGR piglets were fed control TPN (TPN-IUGR) as a fourth group. After 2 wk of treatment, all pigs were then fed a standard solid diet. At 8 mo old, central venous catheters were implanted to conduct postprandial fat tolerance tests.

Feeding TPN in the neonatal period led to dyslipidemia in adulthood, as indicated by higher postprandial triglyceride (TG) levels in TPN-control (P < 0.05), compared with SowFed. IUGR piglets were particularly sensitive to neonatal TPN feeding, as TPN-IUGR piglets developed obesity and dyslipidemia in adulthood, as indicated by greater backfat thickness (P < 0.05), higher liver TG (P < 0.05), slower postprandial TG clearance (P < 0.05), and elevated fasting plasma nonhigh-density lipoprotein-cholesterol (P < 0.01), and nonesterified fatty acids (P < 0.001), compared with TPN-control.

Feeding TPN in early life increases the risk of developing dyslipidemia in adulthood, especially in IUGR neonates; however, methyl nutrient supplementation to TPN did not prevent TPN-induced changes in lipid metabolism.

Overconsumption of sucrose or fat is widely acknowledged as a prominent feature of unhealthy dietary patterns. Both factors commonly co-occur and are recognized as hallmarks of the Western diet, which is an important contributor to non-communicative diseases. In this study, we investigated the hazards of high sucrose or fat intake, either alone or in combination. Wistar rats were divided into four groups and fed a control starch diet, high-sucrose diet, high-fat diet, or high-sucrose/fat diet for 30 days. High fat intake increased body weight and visceral and subcutaneous adipose tissue weights. Both high-sucrose and -fat diets were associated with increased plasma triglyceride and glucose levels, and high sucrose also elevated plasma cholesterol levels. The combination of high sucrose and fat synergistically elevated plasma triglyceride levels. The high-sucrose diet increased liver weight and hepatic total lipid and triglyceride levels, whereas this increase was suppressed by the high-fat diet. The high sucrose increased the mRNA levels of hepatic genes involved in fatty acid synthesis and transport (ACLY, ACACA, FAS, ELOVL6, SCD1, SREBP1, and CD36), whereas the high fat suppressed the high sucrose-induced expression of these genes. We observed that high sucrose and fat contents differently exerted their effects on hyperlipidemia and fatty liver. Furthermore, high fat aggravated hyperlipidemia and suppressed fatty liver induced by high sucrose.

The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease.

Triple Negative Breast cancer is an aggressive breast cancer subtype. It has a more aggressive clinical course, an earlier age of onset, a larger propensity for metastasis, and worse clinical outcomes as evidenced by a higher risk of recurrence and a shorter survival rate. Currently, the primary options for TNBC treatment are surgery, radiation, and chemotherapy. These treatments however remain ineffective due to recurrence. However, given that p53 mutations have been identified in more than 60-88 % of TNBC, translating p53 into the clinical situation is particularly important in TNBC. In this study, we screened and evaluated the therapeutic potential of cryptolepine (CRP) in TNBC in-vitro models being an anti-malarial drug it could be repurposed as an anti-cancer therapeutic targeting TNBC. Moreover, the cytotoxicity activity of cryptolepine to TNBC cells and a detailed anti-tumor mechanism in mutant P53 has not been reported before.

MTT assays were used to examine the cytotoxicity and cell viability activity of Cryptolepine in TNBC, non-TNBC T47D and MCF-7 and non-malignant MCF10A cells. Scratch wound and clonogenic assay was used to evaluate the cryptolepine's effect on migration and colony forming ability of TNBC cells. Flow cytometry, MMP and DAPI was used to assess cell cycle arrest and cell apoptosis mechanism. The expression of proteins was detected by western blots. The differential expression of RNAs was evaluated by RT-PCR and the interaction between P53 and drug was evaluated computationally using in-silico approach and in-vitro using ChIP assay.

In this study, we found that cryptolepine has more preferential cytotoxicity in TNBC than non-TNBC cells. Notably, our studies revealed the mechanism by which cryptolepine induces intrinsic apoptosis and inhibit migration, colony formation ability, induce cell cycle arrest by inducing conformational change in the mutant P53 thereby increasing its DNA binding ability, hence activating its tumor suppressing potential significantly.

Our study revealed that CRP significantly reduced the proliferation, migration and colony forming ability of TNBC cells lines. Moreover, it was revealed that CRP induces cell cycle arrest and apoptosis by activating mutant P53 and enhancing its DNA binding ability to induce its tumor suppressing ability.

Preeclampsia (PE) is a complex multisystem disease characterized by hypertension of sudden onset (>20 weeks' gestation) coupled with the presence of at least one additional complication, such as proteinuria, maternal organ dysfunction, or uteroplacental dysfunction. Hypertensive states during pregnancy carry life-threatening risks for both mother and baby. The pathogenesis of PE develops due to a dysfunctional placenta with aberrant architecture that releases factors contributing to endothelial dysfunction, an antiangiogenic state, increased oxidative stress, and maternal inflammatory responses. Previous studies have shown a correlation between grade 3 placental calcifications and an elevated risk of developing PE at term. However, little is known about the molecular pathways leading to placental calcification. In this work, we studied the gene and protein expression of c-Jun N-terminal kinase (JNK), Runt-related transcription factor 2 (RUNX2), osteocalcin (OSC), osteopontin (OSP), pigment epithelium-derived factor (PEDF), MSX-2/HOX8, SOX-9, WNT-1, and β-catenin in placental tissue from women with late-onset PE (LO-PE). In addition, we employed von Kossa staining to detect mineral deposits in placental tissues. Our results show a significant increase of all these components in placentas from women with LO-PE. Therefore, our study suggests that LO-PE may be associated with the activation of molecular pathways of placental calcification. These results could be the starting point for future research to describe the molecular mechanisms that promote placental calcification in PE and the development of therapeutic strategies directed against it.