The novel small molecular BH3 mimetics SM3 and its regulation of cell apoptosis and autophagy.

Bcl-2 family proteins play an important role in regulation of the cell survival and death. The inhibition of the anti-apoptotic proteins of Bcl-2 family leads to the apoptosis of cancer. BH3 mimetics have been developed targeting anti-apoptotic proteins of Bcl-2 family as small molecular drugs. It has been proved that BH3 mimetics has effect on apoptosis and proliferation in leukemia and some of them has been used in phase one or two clinical trials. Besides, with the development of the research on autophagic cell death, the antagonism and the synergism of autophagy and apoptosis is significant in cell death. As a hub of these two pathways of cell death, Bcl-2 protein is a potential target in basic research and clinical applications. In our studies, we found 32 potential BH3 mimetics compounds from 140,000 small molecular compounds via pharmacophore-based virtual screening. Furthermore, we demonstrated SM3, one of the 32 potential BH3 mimetics, induced autophagy and apoptosis simultaneously in dose-time dependence in A549 cell. SM3 induced apoptosis by intrinsic apoptosis pathway and induced autophagy by weakening the interaction between Beclin-1 and Bcl-2 complex. We wish to provide evidences and clues for the structural optimizing and further study of new compounds in the future.

Cortisol modulates inflammatory responses in LPS-stimulated RAW264.7 cells via the NF-κB and MAPK pathways.

BACKGROUND: The uteruses of most dairy cattle are easily infected by bacteria, especially gram-negative bacteria, following parturition. Macrophages are important cells of the immune system and play a critical role in the inflammatory response. In addition, cortisol levels become significantly increased due to the stress of parturition in dairy cattle, and cortisol is among the most widely used and effective therapies for many inflammatory diseases. In this study, we assessed the anti-inflammatory effects and potential molecular mechanisms of cortisol using a Lipopolysaccharide (LPS)-induced RAW264.7 macrophage cell line. RESULTS: Cortisol significantly suppressed the production of prostaglandin E2 (PGE2) and decreased the gene and protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, cortisol inhibited the mRNA expression of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) and decreased IL-1ß secretion in an LPS-treated RAW264.7 macrophage cell line. Moreover, we found that cortisol suppressed nuclear factor-kappa B (NF-κB) signaling in RAW264.7 macrophages stimulated with LPS. This suppression was mediated by the inhibition of IκBα degradation and NF-κB p65 phosphorylation. In addition, cortisol also suppressed the phosphorylation of mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). CONCLUSIONS: These results suggest that high cortisol levels can attenuate LPS-induced inflammatory responses in the RAW264.7 macrophage cell line by regulating the NF-κB and MAPK signaling pathways.

Enoxaparin sensitizes human non-small-cell lung carcinomas to gefitinib by inhibiting DOCK1 expression, vimentin phosphorylation, and Akt activation.

Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation.

Effects of natural sound exposure on health recovery: A systematic review and meta-analysis.

Listening to natural sounds, both live and recorded, in either a natural or built environment is considered natural sound exposure (NSE). Sound is closely related to daily life, and research on the restorative effects of natural sounds is expanding. However, there is a lack of quantitative and comprehensive analysis on the impact of NSE on health recovery. This study systematically reviewed and conducted a meta-analysis on the impact of NSE on health recovery. Fifteen studies (1285 participants) were selected for the meta-analysis out of the 1157 literatures about the recovery of the NSE, searched from the Web of Science and Science Direct. The results indicate that NSE has certain positive effects: (a) In terms of emotional changes, NSE significantly reduces anxiety as measured by both the Visual Analog Scale (VAS) -2.31 (95 % CI -2.83, -1.79) and the State Anxiety Inventory (SAI) -12.22 (95 % CI -22.46, -1.98). (b) In terms of physiological reaction, NSE resulted in reduced heart rate (HR) -5.46 (95 % CI -9.62, -1.31), systolic blood pressure (SBP) -11.74 (95 % CI -15.51, -7.97), diastolic blood pressure (DBP) -13.98 (95 % CI -24.96, -2.99) and respiratory rate (RR) -1.58 (95 % CI -3.06, -0.10). (c) While the potential for restoration of cognitive performance by NSE was found, no consistent conclusions have been reached yet. However, there was significant heterogeneity between studies, primarily attributed to variations in study populations and methodologies. Because of the limited literature, we did not conduct subgroup analysis and meta-regression analysis. It is recommended that future studies address this heterogeneity by including more and higher-quality literature and employing rigorous methodologies to establish a robust foundation for evidence-based medicine. This will be of great significance for the application natural sounds in landscape planning and medical rehabilitation environments, and has the potential to promote improvements in public health.

Effect of ultra-high pressure heat-assisted technology combined with L-cysteine on the color of ready-to-eat shrimp during storage.

This study used ultra-high pressure processing (HPP) heat-assisted technology combined with L-cysteine (L-cys) to process ready-to-eat (RTE) shrimp. Subsequently, the effects of physical field and chemical modifications on the color of RTE shrimp were studied. The results showed that the RTE shrimp treated with HPP-Heat-L-cys showed better performance in terms of brightness value (65.25) and astaxanthin (AST) content (0.71 µg/g) during storage, maintaining the original color of RTE shrimp effectively. In addition, it was observed that the application of HPP-Heat-L-cys significantly delayed phenol oxidation, lipid oxidation, and Maillard reaction compared with traditional HPP or heat treatments. Specifically, the total phenolic content of RTE shrimp treated with HPP-Heat-L-cys was higher than that of other samples, but the TBARS and browning index were lower. Furthermore, HPP-Heat-L-cys could delay the production of dark products (such as 2-methylanthraquinone, p-benzoquinone, lipofuscin and melanin), ultimately safeguarding the color stability of RTE shrimp during storage.

Nanocellulose from agro-industrial wastes: A review on sources, production, applications, and current challenges.

Significant volumes of agricultural and industrial waste are produced annually. With the global focus shifting towards sustainable and environmentally friendly practices, there is growing emphasis on recycling and utilizing materials derived from such waste, such as cellulose and lignin. In response to this imperative situation, nanocellulose materials have surfaced attracting heightened attention and research interest owing to their superior properties in terms of strength, stiffness, biodegradability, and water resistance. The current manuscript provided a comprehensive review encompassing the resources of nanocellulose, detailed pretreatment and extraction methods, and present applications of nanocellulose. More importantly, it highlighted the challenges related to its processing and utilization, along with potential solutions. After evaluating the benefits and drawbacks of different methods for producing nanocellulose, ultrasound combined with acid hydrolysis emerges as the most promising approach for large-scale production. While nanocellulose has established applications in water treatment, its potential within the food industry appears even more encouraging. Despite the numerous potential applications across various sectors, challenges persist regarding its modification, characterization, industrial-scale manufacturing, and regulatory policies. Overcoming these obstacles requires the development of new technologies and assessment tools aligned with policy. In essence, nanocellulose presents itself as an eco-friendly material with extensive application possibilities, prompting the need for additional research into its extraction, application suitability, and performance enhancement. This review focused on the wide application scenarios of nanocellulose, the challenges of nanocellulose application, and the possible solutions.

Molecular mechanism of color deepening of ready-to-eat shrimp during storage.

Color deepening occurs during storage of ready-to-eat (RTE) shrimps, which seriously affects their marketing cycle. This study investigated the molecular mechanisms of color deterioration in RTE shrimps during accelerated storage, shedding light on the pattern of change in colored products and content. The findings revealed significant occurrences of phenolic oxidation, lipid oxidation, and Maillard browning reactions during accelerated storage. Qualitative and quantitative analyses were conducted on the colored products resulting from these chemical reactions. Multivariate mathematical models were employed to analyze the phenolic oxidation products (2-methylanthraquinone and p-benzoquinone), lipid oxidation products (lipofuscin-like pigments and hydrophobic pyrroles), and Maillard browning products (pyrazines and melanoidins). These products were identified as the main contributors to the deepening of the color of RTE shrimps during storage. The outcomes of this research could enhance our understanding of the color change mechanism in thermally processed marine foods, providing valuable insights for quality maintenance and industrial advancement.

Investigation of isomerization and oxidation of astaxanthin in ready-to-eat Litopenaeus vannamei during accelerated storage.

Astaxanthin (AST), the natural pigment in Litopenaeus vannamei, is susceptible to oxidation and isomerization, leading to the fading of the orange-red color in ready-to-eat (RTE) shrimps. This study specifically investigated the changes mechanism in AST content, including geometric and stereoisomers, as well as oxidation degradation, throughout the storage process of RTE shrimps. The results showed that the total amount of AST decreased by 46.76 % after 45 days of storage at 40 °C. The levels of geometric isomers (all-E, 9-Z, 13-Z) and stereoisomers (3S,3'S, 3S,3'R, 3R,3'R) gradually decreased over time. Notably, 9-Z and 3S,3'S isomers, known for their strong antioxidant activity, were reduced by 83.57 % and 61.64 % respectively. Additionally, AST underwent oxidative degradation, forming short-chain compounds (astaxanthinal or astaxanthinone), with the main products being Apo-14'-astaxanthinal and Apo-7-astaxanthinone DHA ester. These findings provide a theoretical foundation for further research on the degradation mechanism of AST, and offer valuable insights into the color protection of RTE shrimps.

Effects of calcium chloride on the gelling and digestive characteristics of myofibrillar protein in Litopenaeus vannamei.

In this study, the effects of CaCl2 (0, 25, 50, 75, and 100 mM) on the gelling and digestive properties of the myofibrillar protein (MP) in Litopenaeus vannamei were investigated. The results showed that increasing CaCl2 concentration led to changes in the tertiary structure of MP. Specifically, compared with the control group, a 64.31 % increase in surface hydrophobicity and a 45.90 % decrease in the sulfhydryl group were observed after 100 mM CaCl2 treatment. Correspondingly, the water holding capacity and strength of the MP gel increased by 24.46 % and 55.99 %, respectively. These changes were positively correlated with the rheological properties, microstructure pore size, and content of non-flowable water. The mechanical properties of MP gel were improved, and the microstructure became more compact with the increase in CaCl2 concentration. Furthermore, the particle size of the digested MP gels decreased in the presence of CaCl2, which improved the digestion characteristics of MP gels.

Plexin B3 guides axons to cross the midline in vivo.

During the development of neural circuits, axons are guided by a variety of molecular cues to navigate through the brain and establish precise connections with correct partners at the right time and place. Many axon guidance cues have been identified and they play pleiotropic roles in not only axon guidance but also axon fasciculation, axon pruning, and synaptogenesis as well as cell migration, angiogenesis, and bone formation. In search of receptors for Sema3E in axon guidance, we unexpectedly found that Plexin B3 is highly expressed in retinal ganglion cells of zebrafish embryos when retinal axons are crossing the midline to form the chiasm. Plexin B3 has been characterized to be related to neurodevelopmental disorders. However, the investigation of its pathological mechanisms is hampered by the lack of appropriate animal model. We provide evidence that Plexin B3 is critical for axon guidance in vivo. Plexin B3 might function as a receptor for Sema3E while Neuropilin1 could be a co-receptor. The intracellular domain of Plexin B3 is required for Semaphorin signaling transduction. Our data suggest that zebrafish could be an ideal animal model for investigating the role and mechanisms of Sema3E and Plexin B3 in vivo.

Research Progress in Pharmacological Mechanisms, Structure-Activity Relationship and Synthesis of Sartans.

The sartans are a new class of antihypertensive drugs as angiotensin II receptor blockers which possess plenty of advantages in treating hypertension and related pathologies. This review describes the clinical treatment, side effects, and potential therapeutic effects of sartans from 1995 to date. The synthesis, structural-activity and molecular docking with Angiotensin Type 1 receptor of imidazole derivatives, benzimidazole derivatives and other compounds are also described. With a clear Structure-Activity Relationship and abundant pharmacological effects, some types of novel Angiotensin Type 1 receptor antagonists are emerging gradually for further research in the meantime.

Identification of Two Subsets of Subcompartment A1 Associated with High Transcriptional Activity and Frequent Loop Extrusion.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Repeated Systemic Dosing of Adeno-Associated Virus Vectors in Immunocompetent Mice After Blockade of T Cell Costimulatory Pathways.

Neutralizing antibodies (NAbs) strongly limit adeno-associated virus (AAV) vector transduction and repeated administration. Previous studies have shown that NAbs induced by AAVs are associated with T and B cell activation and that the B7/CD28 and CD40/CD40L costimulation signaling pathways are involved. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD40 are vital molecules that participate in the costimulatory pathway. In this study, we evaluated CTLA4-Ig and CD40-Ig immunosuppreve efficacies through AAV and investigated their effects on the feasibility for multiple systemic administrations of AAV vectors. The results showed that a single administration of AAV vector carrying either CTLA4-Ig alone or with CD40-Ig could greatly reduce the level of NAbs. An AAV serotype-specific immune tolerance could be successfully established, which enabled repeated, that is, second and third, systemic administration of AAV vectors in the same mice. A combination of CTLA4-Ig and CD40-Ig delivered via AAV vectors significantly inhibited T and B cell activations without affecting the immune response to the total immunoglobulin G production and cytokines. Interestingly, exogenous gene expression significantly improved after multiple administrations of AAV vector in vivo. Our study generates a reliable and effective method for repeated dosing of AAV vectors that is needed on gene therapy.

[Study on the Therapeutic Effect of Lenalidomide on Hemophilic Arthropathy].

OBJECTIVE: To explore the effect of lenalidomide on human fibroblast-like synovial cells (HFLS) and the therapeutic efficacy on hemophilic arthropathy in hemophilia A mice model. METHODS: In vitro, to remodel the inflammatory environment of synovial tissue after hemorrhage, ferric citrate and recombinant TNF-α were added into the cell culture medium of HFLS. Cell Counting Kit-8 (CCK-8), Enzyme-linked immunosorbent assay (ELISA), Quantitative Real-time PCR (RT-qPCR) and flow cytometry were employed for detection of the effects of lenalidomide on the proliferation ability, pro-inflammatory cytokines release and apoptosis of HFLS cells. In vivo, hemophilia arthropathy was remodeled in hemophilia A mice by induction of hemarthrosis. A series of doses of lenalidomide (0.1, 0.3 and 1.0 g/kg) was administrated intra-articularly. Tissues of knee joints were collected on the 14th day after administration, and the protective effect of lenalidomide on arthritis in hemophilia A mice were evaluated by RT-qPCR and histological grading. RESULTS: In vitro, compared with the untreated control group, lenalidomide could significantly inhibit the proliferation of HFLS cells (P<0.05), and the effect was the most significant when the concentration was 0.01 µmol/L (P<0.001). Compared with the control group, lenalidomide could significantly inhibit the expression levels of TNF-α, IL-1ß, IL-6 and IFN-γ in HFLS cells (P<0.05). The flow cytometry results showed that lenalidomide could enhance the apoptotis of HFLS cells (P<0.05). The results of RT-qPCR showed that lenalidomide could significantly reduce the mRNA expression levels of TNF-α, IL-1ß, IL-6,MCP-1 and VEGF in the joint tissues (P<0.05). Histological results showed that compared with the injured group, lenalidomide could significantly reduce the pathological sequela after hemarthrosis induction, e.g. synovial thickening and neo-angiogenesis in the synovium. The protection displayed a dose-response pattern roughly. CONCLUSION: In vitro, lenalidomide can inhibit the proliferation of HFLS cells, promote their apoptosis, and inhibit the expression of pro-inflammatory cytokines. In vivo, lenalidomide can significantly decrease the expression of pro-inflammatory cytokines in the joints of mice, and prevent the development of inflammation and neo-angiogenesis. The results provide a theoretical and experimental basis for the clinical application of lenalidomide in the treatment of hemophilic arthropathy.

SCSit: A high-efficiency preprocessing tool for single-cell sequencing data from SPLiT-seq.

SPLiT-seq provides a low-cost platform to generate single-cell data by labeling the cellular origin of RNA through four rounds of combinatorial barcoding. However, an automatic and rapid method for preprocessing and classifying single-cell sequencing (SCS) data from SPLiT-seq, which directly identified and labeled combinatorial barcoding reads and distinguished special cell sequencing data, is currently lacking. Here, we develop a high-efficiency preprocessing tool for single-cell sequencing data from SPLiT-seq (SCSit), which can directly identify combinatorial barcodes and UMI of cell types and obtain more labeled reads, and remarkably enhance the retained data from SCS due to the exact alignment of insertion and deletion. Compared with the original method used in SPLiT-seq, the consistency of identified reads from SCSit increases to 97%, and mapped reads are twice than the original. Furthermore, the runtime of SCSit is less than 10% of the original. It can accurately and rapidly analyze SPLiT-seq raw data and obtain labeled reads, as well as effectively improve the single-cell data from SPLiT-seq platform. The data and source of SCSit are available on the GitHub website https://github.com/shang-qian/SCSit.

Cortisol inhibits NF-κB and MAPK pathways in LPS activated bovine endometrial epithelial cells.

The bovine uterus is subject to infection after calving, which may lead to endometritis. Elevated cortisol levels have been observed in postpartum cattle. However, the role of cortisol in the inflammatory response of the uterus has not been reported. The aim of this study was to investigate the anti-inflammatory effects of cortisol on lipopolysaccharide (LPS)-induced primary bovine endometrial epithelial cells (BEECs). BEECs were treated with various concentrations of cortisol (5, 15 and 30 ng/mL) in the presence of LPS. The mRNA expression of TLR4 and proinflammatory cytokines was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blotting and immunofluorescence. Cortisol induced the down-regulation of the mRNA expression of toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). Cortisol inhibited the activity of nuclear factor-κB (NF-κB) via blocking the phosphorylation and degradation of IκB. Cortisol suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK1/2), p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). These results demonstrated that cortisol may exert its anti-inflammatory actions by regulating NF-κB activation and MAPK phosphorylation.

Comparative Brain and Central Nervous System Tumor Incidence and Survival between the United States and Taiwan Based on Population-Based Registry.

PURPOSE: Reasons for worldwide variability in the burden of primary malignant brain and central nervous system (CNS) tumors remain unclear. This study compares the incidence and survival of malignant brain and CNS tumors by selected histologic types between the United States (US) and Taiwan. METHODS: Data from 2002 to 2010 were selected from two population-based cancer registries for primary malignant brain and CNS tumors: the Central Brain Tumor Registry of the United States and the Taiwan Cancer Registry. Two registries had similar process of collecting patients with malignant brain tumor, and the quality of two registries was comparative. The age-adjusted incidence rate (IR), IR ratio, and survival by histological types, age, and gender were used to study regional differences. RESULTS: The overall age-adjusted IRs were 5.91 per 100,000 in the US and 2.68 per 100,000 in Taiwan. The most common histologic type for both countries was glioblastoma (GBM) with a 12.9% higher proportion in the US than in Taiwan. GBM had the lowest survival rate of any histology in both countries (US 1-year survival rate = 37.5%; Taiwan 1-year survival rate = 50.3%). The second largest group was astrocytoma, excluding GBM and anaplastic astrocytoma, with the distribution being slightly higher in Taiwan than in the US. CONCLUSION: Our findings revealed differences by histological type and grade of primary malignant brain and CNS tumors between two sites.

Layered signaling regulatory networks analysis of gene expression involved in malignant tumorigenesis of non-resolving ulcerative colitis via integration of cross-study microarray profiles.

BACKGROUND: Ulcerative colitis (UC) was the most frequently diagnosed inflammatory bowel disease (IBD) and closely linked to colorectal carcinogenesis. By far, the underlying mechanisms associated with the disease are still unclear. With the increasing accumulation of microarray gene expression profiles, it is profitable to gain a systematic perspective based on gene regulatory networks to better elucidate the roles of genes associated with disorders. However, a major challenge for microarray data analysis is the integration of multiple-studies generated by different groups. METHODOLOGY/PRINCIPAL FINDINGS: In this study, firstly, we modeled a signaling regulatory network associated with colorectal cancer (CRC) initiation via integration of cross-study microarray expression data sets using Empirical Bayes (EB) algorithm. Secondly, a manually curated human cancer signaling map was established via comprehensive retrieval of the publicly available repositories. Finally, the co-differently-expressed genes were manually curated to portray the layered signaling regulatory networks. RESULTS: Overall, the remodeled signaling regulatory networks were separated into four major layers including extracellular, membrane, cytoplasm and nucleus, which led to the identification of five core biological processes and four signaling pathways associated with colorectal carcinogenesis. As a result, our biological interpretation highlighted the importance of EGF/EGFR signaling pathway, EPO signaling pathway, T cell signal transduction and members of the BCR signaling pathway, which were responsible for the malignant transition of CRC from the benign UC to the aggressive one. CONCLUSIONS: The present study illustrated a standardized normalization approach for cross-study microarray expression data sets. Our model for signaling networks construction was based on the experimentally-supported interaction and microarray co-expression modeling. Pathway-based signaling regulatory networks analysis sketched a directive insight into colorectal carcinogenesis, which was of significant importance to monitor disease progression and improve therapeutic interventions.

Epididymal sperm count.

Making Epididymal Sperm Counts (Yefan Wang, TherImmune Research Corporation, Gaithersberg, Maryland). Epididymal sperm counts are a widely used, simple and sensitive method of assessing the effects of male reproductive toxicants on the epididymal and/or testicular site of action. After careful dissection of the tissues and further processing, the sperm suspensions are counted using a hemacytometer and analyzed for effect.