HDAC Inhibitors Alleviate Uric Acid-Induced Vascular Endothelial Cell Injury by Way of the HDAC6/FGF21/PI3K/AKT Pathway.

ABSTRACT: Uric acid (UA) accumulation triggers endothelial dysfunction, oxidative stress, and inflammation. Histone deacetylase (HDAC) plays a vital role in regulating the pathological processes of various diseases. However, the influence of HDAC inhibitor on UA-induced vascular endothelial cell injury (VECI) remains undefined. Hence, this study aimed to investigate the effect of HDACs inhibition on UA-induced vascular endothelial cell dysfunction and its detailed mechanism. UA was used to induce human umbilical vein endothelial cell (HUVEC) injury. Meanwhile, potassium oxonate-induced and hypoxanthine-induced hyperuricemia mouse models were also constructed. A broad-spectrum HDAC inhibitor trichostatin A (TSA) or selective HDAC6 inhibitor TubastatinA (TubA) was given to HUVECs or mice to determine whether HDACs can affect UA-induced VECI. The results showed pretreatment of HUVECs with TSA or HDAC6 knockdown-attenuated UA-induced VECI and increased FGF21 expression and phosphorylation of AKT, eNOS, and FoxO3a. These effects could be reversed by FGF21 knockdown. In vivo, both TSA and TubA reduced inflammation and tissue injury while increased FGF21 expression and phosphorylation of AKT, eNOS, and FoxO3a in the aortic and renal tissues of hyperuricemia mice. Therefore, HDACs, especially HDAC6 inhibitor, alleviated UA-induced VECI through upregulating FGF21 expression and then activating the PI3K/AKT pathway. This suggests that HDAC6 may serve as a novel therapeutic target for treating UA-induced endothelial dysfunction.

The prodigiosin change on the surface of Serratia marcescens detected by flow cytometry.

The potential of flow cytometry for the study of changes in prodigiosin on the cell surface of Serratia marcescens is of academic and practical interest. This is because S. marcescens can produce prodigiosin, a secondary metabolite, with potential use as a cancer-cell inhibitor. In this study, three groups of bacterial cultures with different carbon sources were compared, and the effect of the addition of cAMP to the sucrose-based culture was studied. Both cellular morphology and DNA content were detected by flow cytometry, rendering a broad description of the bacterial behavior. It is the first use of flow cytometry to investigate the dynamics of prodigiosin on the surface of S. marcescens during growth in different media. The fluorescence intensity is related to the DNA content, the forward-scattered light is related to cell volume, and the side-scattered light is related to the surface morphology, especially the surface prodigiosin. These may contribute to the potential development of a bacterial metabolic monitoring strategy using both DNA content analysis and bacterial morphology based on flow cytometry technique.

New Organoselenium (NSAIDs-Selenourea and Isoselenocyanate) Derivatives as Potential Antiproliferative Agents: Synthesis, Biological Evaluation and in Silico Calculations.

In this study, we report on the synthesis of new organoselenium derivatives, including nonsteroidal anti-inflammatory drugs (NSAIDs) scaffolds and Se functionalities (isoselenocyanate and selenourea), which were evaluated against four types of cancer cell line: SW480 (human colon adenocarcinoma cells), HeLa (human cervical cancer cells), A549 (human lung carcinoma cells), MCF-7 (human breast adenocarcinoma cells). Among these compounds, most of the investigated compounds reduced the viability of different cancer cell lines. The most promising compound 6b showed IC50 values under 10 µM against the four cancer cell lines, particularly to HeLa and MCF-7, with IC50 values of 2.3 and 2.5 µM, respectively. Furthermore, two compounds, 6b and 6f, were selected to investigate their ability to induce apoptosis in MCF-7 cells via modulation of the expression of anti-apoptotic Bcl-2 protein, pro-inflammatory cytokines (IL-2) and proapoptotic caspase-3 protein. The redox properties of the NSAIDs-Se derivatives were conducted by 2, 2-didiphenyl-1-picrylhydrazyl (DPPH), bleomycin-dependent DNA damage and glutathione peroxidase (GPx)-like assays. Finally, a molecular docking study revealed that an interaction with the active site of thioredoxin reductase 1 (TrxR1) predicted the antiproliferative activity of the synthesized candidates. Overall, these results could serve as a promising launch point for further designs of NSAIDs-Se derivatives as potential antiproliferative agents.

Baculovirus transit through insect cell membranes: A mechanistic approach.

Baculovirus systems are used for various purposes, but the kinetics of the infection process is not fully understood yet. We investigated the dynamics of virion movement from a medium toward the interior of insect cells and established a mechanistic model that shows an excellent fit to experimental results. It also makes possible a description of the viral dynamics on the cell surface. A novel measurement method was used to distinguish between infected cells that carry virions on their surfaces, cells that carry virions in their interior, and those carrying virions both inside and on their surface. The maximum number of virions carried by a cell: 55 viruses/cell, and the time required for viral internalization, 0.8 h , are reported. This information is particularly useful for assessing the infection efficacy and the required number of virions needed to infect a given cell population. Although our model specifically concerns the infection process of Sf9 insect cells by baculovirus, it describes general features of viral infection. Some of the model features may eventually be applicable in the studies towards palliation of the COVID-19 outbreak.

Synthesis and Potential Anticancer Activity of Some Novel Selenocyanates and Diselenides.

In the present study, twenty-four selenocyanate and diselenide compounds were synthesized and characterized, and their anticancer activities against the human cancer cell lines Caco2, BGC-823, MCF-7 and PC-3 were determined. Interestingly, most of the new compounds were active in reducing the viability of different cancer cell lines. Two compounds exhibited higher promising activities than other derivatives. The most active compound showed the least IC50 values against the four cancer cell lines, particularly to PC-3 with IC50 values below 5 µm. Two compounds were selected to monitor the expression levels of Bcl-2, IL-2 and caspase-3 molecular biomarkers. Interestingly, the two compounds downregulated the Bcl-2 expression levels and upregulated the expression of IL-2 and caspase-3 in PC-3 cells compared to untreated cells. Moreover, most of the synthesized organoselenides exhibited good Gpx-like activities comparable to ebselen. These results appear that introduction of selenocyanate (-SeCN) or diselenides (-Se-Se-) moiety to some carboxy derivatives could serve as a promising launch point for the further design of this type of organic selenium anticancer agent.

Venomous snakebites in children: a 10 year experience in South China.

INTRODUCTION: Many studies have focused on snakebites in adults, but very few have described snakebites in children. METHODS: We reviewed the clinical characteristics and outcomes of children with venomous snakebites aged less than 15 years who presented to a regional medical centre in South China from January 2013 to December 2022. RESULTS: A total of 69 envenomed patients were analyzed in our study; 42 (60.9 per cent) patients were male, and 59 (85.5 per cent) reported lower limb bites. Most bites (89.8 per cent) occurred between April and October. Twenty-seven patients received first aid management, and 47 required admission to the general ward. Antivenom was administered to 58 patients, glucocorticoids to 43 patients, antibiotics to 48 patients, and tetanus antitoxin to 40 patients. No fatalities were reported. The most common snake identified was Trimeresurus albolabris. Four were classified as dry bites, 15 as mild, 43 as moderate, and seven as severe. The most common local signs were pain and swelling, while the most common systemic effects were haematological complications. Patients with high severity scores had significantly higher lactate dehydrogenase activities, creatine kinase isoenzyme activities, aspartate aminotransferase activities, D-dimer concentrations, prothrombin times and lower fibrinogen concentrations. In a receiver operating characteristic curve analysis of the values with the highest Youden index, the following cut-offs proved significant: lactate dehydrogenase activity > 248.1 U/L, creatine kinase isoenzyme activities > 17.5 U/L, fibrinogen concentration < 1,455 mg/L, D-dimer concentration > 437.0 µg/L, aspartate aminotransferase activity > 26.1 U/L, and prothrombin time > 15.2 seconds. DISCUSSION: This study provides insight into the epidemiology, clinical profile, and management of snakebites in children. Data from the present study were compared with those from our previous adult study. Limitations include that 50.7 per cent of our snakebites were attributed to Trimeresurus albolabris. Therefore, the results of our study may not be generalizable to all snakebites. CONCLUSION: The clinical symptoms were more severe in children than in adults in our previous study. Even though there were no fatalities, close monitoring should be performed to detect haematological and other potentially fatal complications promptly.

The Role of BmTMED6 in Female Reproduction in Silkworm, Bombyx mori.

Transmembrane emp24 domain (TMED) proteins have been extensively studied in mammalian embryonic development, immune regulation, and signal transduction. However, their role in insects, apart from Drosophila melanogaster, remains largely unexplored. Our previous study demonstrated the abundant expression of BmTMED6 across all stages and tissues of the silkworm. In this study, we investigate the function of BmTMED6 in reproduction. We observe significant differences in the expression of BmTMED6 between male and female silkworms, particularly in the head and fatboby, during the larval stage. Furthermore, qRT-PCR and WB analysis reveal substantial variation in BmTMED6 levels in the ovaries during pupal development, suggesting a potential association with silkworm female reproduction. We find that reducing TMED6 expression significantly decreases the number of eggs laid by female moths, leading to an accumulation of unlaid eggs in the abdomen. Moreover, downregulation of BmTMED6 leads to a decrease in the expression of BmDop2R1 and BmDop2R2, while overexpression of BmTMED6 in vitro has the opposite effect. These indicate that BmTMED6 plays a role in oviposition in female moths, potentially through the dopamine signaling pathway. This study provides a new regulatory mechanism for female reproduction in insects.

The Spatio-Temporal Expression Profiles of Silkworm Pseudogenes Provide Valuable Insights into Their Biological Roles.

Background: Pseudogenes are sequences that have lost the ability to transcribe RNA molecules or encode truncated but possibly functional proteins. While they were once considered to be meaningless remnants of evolution, recent researches have shown that pseudogenes play important roles in various biological processes. However, the studies of pseudogenes in the silkworm, an important model organism, are limited and have focused on single or only a few specific genes. Objective: To fill these gaps, we present a systematic genome-wide studies of pseudogenes in the silkworm. Methods: We identified the pseudogenes in the silkworm using the silkworm genome assemblies, transcriptome, protein sequences from silkworm and its related species. Then we used transcriptome datasets from 832 RNA-seq analyses to construct spatio-temporal expression profiles for these pseudogenes. Additionally, we identified tissue-specifically expressed and differentially expressed pseudogenes to further understand their characteristics. Finally, the functional roles of pseudogenes as lncRNAs were systematically analyzed. Results: We identified a total of 4410 pseudogenes, which were grouped into 4 groups, including duplications (DUPs), unitary pseudogenes (Unitary), processed pseudogenes (retropseudogenes, RETs), and fragments (FRAGs). The most of pseudogenes in the domestic silkworm were generated before the divergence of wild and domestic silkworm, however, the domestication may also involve in the accumulation of pseudogenes. These pseudogenes were clearly divided into 2 cluster, a highly expressed and a lowly expressed, and the posterior silk gland was the tissue with the most tissue-specific pseudogenes (199), implying these pseudogenes may be involved in the development and function of silkgland. We identified 3299 lncRNAs in these pseudogenes, and the target genes of these lncRNAs in silkworm pseudogenes were enriched in the egg formation and olfactory function. Conclusions: This study replenishes the genome annotations for silkworm, provide valuable insights into the biological roles of pseudogenes. It will also contribute to our understanding of the complex gene regulatory networks in the silkworm and will potentially have implications for other organisms as well.

C/EBP{delta} targets cyclin D1 for proteasome-mediated degradation via induction of CDC27/APC3 expression.

The transcription factor CCAAT/enhancer binding protein delta (C/EBPdelta, CEBPD, NFIL-6beta) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPdelta exerts its effect are largely unknown. Here, we report that C/EBPdelta induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPdelta knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3beta. Silencing of C/EBPdelta, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPdelta, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.

Three-dimensional impact-angle-constrained cooperative guidance strategy against maneuvering target.

In order to improve the multiple-missile cooperative attack capability and penetration capability, this paper investigates two three-dimensional impact-angle-constrained cooperative guidance strategies against maneuvering target for controllable thrust missiles. First, a three-dimensional nonlinear guidance model is established that does not assume small missile lead angles in the guidance process. Second, in the line-of-sight (LOS) direction of the cluster cooperative guidance strategy, the proposed guidance algorithm transforms the simultaneous attack problem into a second-order multiagent consensus problem, which effectively solves the practical problem of low guidance precision provoked by the time-to-go estimation. Then, by combining second-order sliding mode control (SMC) and nonsingular terminal SMC theory, the guidance algorithms in the normal and lateral directions to the LOS are designed, respectively, so that the multi-missile can accurately attack a maneuvering target while satisfying the impact angle constraints. Finally, by utilizing the second-order multiagent consensus tracking control in the leader-following cooperative guidance strategy, a novel leader-following time consistency algorithm is investigated to ensure that the leader and followers can attack the maneuvering target simultaneously. Moreover, the stability of the investigated guidance algorithms is proved mathematically. The effectiveness and superiority of the proposed cooperative guidance strategies are verified by numerical simulations.

IRX2 activated by jumonji domain-containing protein 2A is crucial for cardiac hypertrophy and dysfunction in response to the hypertrophic stimuli.

BACKGROUND: Iroquois homeobox 2 (IRX2) is a member of the Iroquois family whose upregulation has been potentially correlated to cardiac hypertrophy. This work studied the function of IRX2 and its related molecules in hypertrophic cardiomyopathy (HCM). METHODS: A GEO dataset GSE32453 was analyzed to identify aberrantly expressed genes in HCM. Altered expression of IRX2 was induced in mice by lentivirus injection, followed by angiotensin II (Ang II) treatment to induce HCM. The function of IRX2 knockdown in ventricular dysfunction, heart volume and pathological changes in mice, and in surface area, oxidative stress and apoptosis of isolated cardiomyocytes were examined. Binding relationship between jumonji domain-containing protein 2A (JMJD2A) and IRX2 was predicted by online tools and validated. The interaction between JMJD2A and IRX2 in HCM development was examined by joint interventions. RESULTS: IRX2 was highly expressed in heart tissues with HCM. IRX2 knockdown prevented mice from Ang II-induced ventricular dysfunction, cardiac hypertrophy, inflammation and fibrosis in mouse heart, and it decreased the levels of cardiac hypertrophy-related markers, oxidative stress response, and apoptosis of Ang II-treated cardiomyocytes. JMJD2A catalyzed demethylation of H3K9me3 near the IRX2 promoter to activate its transcription. JMJD2A knockdown similarly exerted protective functions against cardiac hypertrophy in vivo and in vitro, but the protection was blocked upon further IRX2 upregulation. IRX2 was found to increase the Wnt/ß-catenin signaling activation. CONCLUSION: This work reports that JMJD2A activates IRX2 transcription and the Wnt/ß-catenin signaling to induce cardiac hypertrophy and dysfunction in HCM.

Systematic identification of smORFs in domestic silkworm (Bombyx mori).

The silkworm (Bombyx mori) is not only an excellent model species, but also an important agricultural economic insect. Taking it as the research object, its advantages of low maintenance cost and no biohazard risks are considered. Small open reading frames (smORFs) are an important class of genomic elements that can produce bioactive peptides. However, the smORFs in silkworm had been poorly identified and studied. To further study the smORFs in silkworm, systematic genome-wide identification is essential. Here, we identified and analyzed smORFs in the silkworm using comprehensive methods. Our results showed that at least 738 highly reliable smORFs were found in B. mori and that 34,401 possible smORFs were partially supported. We also identified some differentially expressed and tissue-specific-expressed smORFs, which may be closely related to the characteristics and functions of the tissues. This article provides a basis for subsequent research on smORFs in silkworm, and also hopes to provide a reference point for future research methods for smORFs in other species.

Long non-coding RNA LEISA promotes progression of lung adenocarcinoma via enhancing interaction between STAT3 and IL-6 promoter.

Long non-coding RNAs (lncRNAs) are emerging as a new class of regulators for a variety of biological processes and have been suggested to play pivotal roles in cancer development and progression. Our current study found that a lncRNA, designated enhancing IL-6/STAT3 signaling activation (LEISA, ENST00000603468), functioned as an oncogenic lncRNA in lung adenocarcinoma (LAD), a major form of non-small cell lung carcinoma, which is one of the most frequently diagnosed malignancies with high morbidity and mortality worldwide, and was involved in the regulation of STAT3 induced IL-6 transcription. Our data showed that LEISA was highly expressed in, and correlated with the clinical progression and prognosis of LAD. Ectopic expression of LEISA promoted the proliferation and suppressed apoptosis of LAD cells in vitro and in vivo. Mechanistically, we demonstrated that LEISA recruited STAT3 to bind the promoter of IL-6 and upregulated IL-6 expression. Taken together, our work identifies LEISA as a potential diagnostic biomarker and therapeutic target for LAD.

Cytochrome P450 monooxygenase genes in the wild silkworm, Bombyx mandarina.

Wild (Bombyx mandarina) and domestic silkworms (B. mori) are good models for investigating insect domestication, as 5000 years of artificial breeding and selection have resulted in significant differences between B. mandarina and B. mori. In this study, we improved the genome assemblies to the chromosome level and updated the protein-coding gene annotations for B. mandarina. Based on this updated genome, we identified 68 cytochrome P450 genes in B. mandarina. The cytochrome P450 repository in B. mandarina is smaller than in B. mori. Certain currently unknown key genes, rather than gene number, are critical for insecticide resistance in B. mandarina, which shows greater resistance to insecticides than B. mori. Based on the physical maps of B. mandarina, we located 66 cytochrome P450s on 18 different chromosomes, and 27 of the cytochrome P450 genes were concentrated into seven clusters. KEGG enrichment analysis of the P450 genes revealed the involvement of cytochrome P450 genes in hormone biosynthesis. Analyses of the silk gland transcriptome identified candidate cytochrome P450 genes (CYP306A) involved in ecdysteroidogenesis and insecticide metabolism in B. mandarina.

An RFC4/Notch1 signaling feedback loop promotes NSCLC metastasis and stemness.

Notch signaling represents a key mechanism mediating cancer metastasis and stemness. To understand how Notch signaling is overactivated to couple tumor metastasis and self-renewal in NSCLC cells, we performed the current study and showed that RFC4, a DNA replication factor amplified in more than 40% of NSCLC tissues, directly binds to the Notch1 intracellular domain (NICD1) to competitively abrogate CDK8/FBXW7-mediated degradation of NICD1. Moreover, RFC4 is a functional transcriptional target gene of Notch1 signaling, forming a positive feedback loop between high RFC4 and NICD1 levels and sustained overactivation of Notch signaling, which not only leads to NSCLC tumorigenicity and metastasis but also confers NSCLC cell resistance to treatment with the clinically tested drug DAPT against NICD1 synthesis. Furthermore, together with our study, analysis of two public datasets involving more than 1500 NSCLC patients showed that RFC4 gene amplification, and high RFC4 and NICD1 levels were tightly correlated with NSCLC metastasis, progression and poor patient prognosis. Therefore, our study characterizes the pivotal roles of the positive feedback loop between RFC4 and NICD1 in coupling NSCLC metastasis and stemness properties and suggests its therapeutic and diagnostic/prognostic potential for NSCLC therapy.

Comparative genome-wide DNA methylation analysis reveals epigenomic differences in response to heat-humidity stress in Bombyx mori.

DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response.

AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex.

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree.

[Inhibitory effects of an antisense PCDGF vector on proliferation and invasion of highly malignant ovarian cancer cells and the related mechanism].

OBJECTIVE: To investigate the inhibitory effects of an antisense PC cell derived growth factor (PCDGF) vector on proliferation and invasion of highly malignant ovarian cancer cell lines Sw626 and A2780 cells, and preliminarily explore the related mechanisms. METHODS: MTT assay and Boyden chamber in vitro invasion assay were employed to detect the changes of proliferation and invasion ability in the Sw626 and A2780 cells transfected with anti-sense PCDGF. The expression levels of cyclin D1 and CDK4 proteins before and after transfection were detected by Western blotting. The effects on the expression and activity of MMP-2 were evaluated by quantitative RT-PCR and zymography, respectively. RESULTS: Comparing with the blank group, the proliferation inhibition rate of the Sw626 and A2780 cells transfected with anti-sense PCDGF was 72.9% and 70.9%, respectively, and the invasion ability was inhibited by 62.9% and 59.0%, respectively. The levels of cyclin D1 and CDK4 protein expression in antisense PCDGF transfected cells were 0.38 +/- 0.08 and 0.37 +/- 0.13, respectively, all significantly lower than 0.84 +/- 0.11 and 0.64 +/- 0.11, respectively, in the blank group (P < 0.01). The MMP-2 mRNA expression level in antisense PCDGF transfected cell group was 0.66 +/- 0.11, not significantly decreased in comparison with 0.89 +/- 0.09 in the blank group (P > 0.05), but the activity of MMP-2 was inhibited significantly. CONCLUSION: The antisense PCDGF vector may inhibit markedly the proliferation and invasion of highly malignant ovarian cancer cells, and partially reverses their malignant phenotype. It seems to be related with down-regulating the expression of cyclin D1 and CDK4 and inhibiting the activity of MMP-2. Our findings indicate that PCDGF may become a new target for antisense gene therapy of ovarian cancer.

Ethyl 4-(4-cyano-phen-yl)-6-methyl-2-thioxo-1,2,3,4-tetra-hydro-pyrimidine-5-carboxyl-ate.

The asymmetric unit of the title compound, C(15)H(15)N(3)O(2)S, contains two independent mol-ecules corresponding to the R and S enanti-omers. The dihydro-pyrimidinone rings adopt a flattened boat conformation. One of the ethyl groups is disordered over two orientations with occupancy factors of 0.700 (7) and 0.300 (7). In the crystal structure, mol-ecules are linked by inter-molecular N-H⋯O hydrogen-bonding inter-actions into one-dimensional chains along the c-axis direction. The chains are further connected by N-H⋯S hydrogen bonds, forming a three-dimensional network.

3-Nitro-benzaldehyde thio-semicarbazone.

The mol-ecule of the title compound, C(8)H(8)N(4)O(2)S, adopts an E configuration about both the C-N bonds. In the crystal structure, adjacent mol-ecules are linked by inter-molecular N-H⋯S hydrogen-bonding inter-actions, forming chains running parallel to the b axis.