BACKGROUND AND OBJECTIVES: The gestational weight gain (GWG) range during mid-to-late pregnancy associated with the lowest combined risk of adverse birth outcomes in women with gestational diabetes mellitus (GDM) remains unclear. This study aims to examine the associations between GWG and adverse birth out-comes among women with GDM. METHODS AND STUDY DESIGN: This study included a cohort of 1,673 pregnant women with GDM. GWG was defined as weight gain from pre-pregnancy to a measurement between 24 and 32 gestational weeks, residualized for gestational age and standardized as z-scores. Multivariable logistic regression models were used to assess associations between GWG z-scores (per 1-SD increase and categories: <-1, -1 to 1 [reference], and ≥1) and small for gestational age (SGA), large for gestational age (LGA), and preterm birth. Restricted cubic splines were fitted to explore nonlinear associations. RESULTS: Each 1-SD higher in GWG z-score was associated with lower odds of SGA (odds ratio [OR], 0.55; 95% confidence interval [CI], 0.43-0.71) and higher odds of LGA (1.48; 1.29-1.70). Compared with the reference group, women with GWG z-scores <-1 had higher odds of SGA and lower odds of LGA, whereas those with z-scores ≥1 showed the opposite pattern. No significant association was observed with preterm birth. Spline analyses indicated that a GWG z-score of approximately -0.07 (equal to 8.2 kg at 28 weeks) was associated with the lowest combined odds of SGA and LGA. CONCLUSIONS: Among women with GDM, both insufficient and excessive mid-to-late pregnancy GWG were associated with adverse size-for-gestational-age outcomes.

PubMed2027

A Conditional Knockout Mutant Approach to Study Essential Genes in Chlamydia in Cell Culture and In Vivo.

Methods in molecular biology (Clifton, N.J.)Samantha K D'Spain, María Eugenia Cortina, R Clayton Bishop, Isabelle Derré

Identification and characterization of essential Chlamydia-specific factors are vital for the generation of effective therapeutics and vaccines. Despite delays in the development of genetic tools for Chlamydia, significant advancements in Chlamydia genetic manipulation have been made over the last decade and a half, now allowing for the targeted interruption or deletion of chromosomal genes. However, because of the obligate intracellular nature of the bacteria and its unique biphasic developmental cycle, it remains challenging to inactivate essential genes and study the function of their encoded proteins. Here, we describe a method to generate conditional knockout Chlamydia mutants by introducing, prior to gene inactivation, a complementation plasmid containing the gene of interest under the control of an anhydrotetracycline (aTc)-inducible promoter. The chromosomal copy of the gene can then be selectively deleted to produce a conditional knockout mutant in which expression of the gene is controlled with aTc. We detail how this conditional knockout mutant can be used to determine if the targeted gene is essential for Chlamydia infection in cell culture and in vivo.

PubMed2027

A Modelling Approach to Resolve Gene Temporal Dynamics in Intestinal Inflammation.

Methods in molecular biology (Clifton, N.J.)Shivam Patel, Brinda Balasubramanian, Paloma Ordóñez-Morán

The intestinal epithelium is maintained by stem cells at the crypt base, however, acute or chronic inflammation can severely disrupt stem cell function and tissue homeostasis. To characterize mucosal inflammation and tissue damage, we evaluated time-course gene expression changes using a dextran sulfate sodium (DSS)-induced mouse model of colitis. By applying normalization, Z-score transformation, and spline curve modeling, we trace the temporal expression dynamics of key intestinal genes. This computational approach offer insights into the molecular responses during inflammation and may help identifying new biomarkers for chronic inflammatory diseases.

PubMed2027

A Straightforward and Reliable Mouse Model of Colorectal Cancer by Orthotopic Transplantation.

Methods in molecular biology (Clifton, N.J.)Cewen Chen, Qiaochu Fu, Chao Gao, Shinya Tanaka, Masamichi Imajo

Colorectal cancer (CRC) is a major malignancy with significant global implications for human health. The development of a reliable and pathologically relevant orthotopic CRC model is essential for advancing our understanding of its molecular mechanisms and for developing more effective therapeutic interventions. However, the construction of such models is fraught with challenges primarily because of the technical complexities involved in the transplantation of CRC cells into the intestinal epithelium. In this chapter, we describe a recently developed method for generating an orthotopic CRC model within the mouse cecal epithelium. This model enables tumor development and progression within a natural tissue microenvironment and facilitates complex interactions between tumor cells and surrounding normal cells, thereby replicating the intratumor heterogeneity of CRC. All procedures in this method are visualized under stereomicroscopic observation, leading to the efficient engraftment of CRC cells and subsequent tumor development. The establishment of this reliable orthotopic CRC model, which mimics tumor development in a more natural microenvironment, offers new opportunities to explore the molecular mechanisms underlying CRC and to assess novel anticancer therapies in pathologically relevant contexts.

PubMed2027

A Tissue Explant Model for Colon Cancer.

Methods in molecular biology (Clifton, N.J.)Justine Epiney, Stefano Gussago, Pénélope Saint-Amour, Joe Khallouf, Teresa Torres, Aurélie Vuagniaux, Nicola Colucci, Fabio Butti, Frédéri…

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide. Recent advancements in cancer research emphasize the importance of personalized medicine, necessitating robust preclinical models that closely mimic the tumor microenvironment. Colorectal cancer organoids have emerged as a pivotal tool in this domain, providing a platform that preserves the cellular heterogeneity and allows high-throughput drug testing. However, organoids do not recapitulate the complexity of the tumor ecosystem, its architecture, and stromal interactions characteristic of native tumors. Here, we describe a model to maintain CRC tissue explants in culture. These explants enable the assessment of therapeutic responses ex vivo. Furthermore, they may offer insights into tumor-immune interactions and facilitate the evaluation of novel immunotherapeutic agents. The continued refinement of CRC tissue explant models promises to enhance our understanding of tumor biology and improve the predictive accuracy of preclinical studies, ultimately contributing to more effective and personalized treatment strategies for colorectal cancer patients.

PubMed2027

AA and Parkinson's Disease.

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

Parkinson's disease (PD) is due to degeneration of dopaminergic neurons in substantia nigra pars compacta. PD neuropathology is characterized by the presence of Lewy bodies that contain aggregated α-synuclein, which has been implicated in a range of interactions with phospholipid membranes and free fatty acids. I propose that α-synuclein interacts with arachidonic acid (AA) to regulate the function of dopaminergic neurons. α-Synuclein sequesters AA, whereas AA induces the formation of oligomers that are alpha-helical; resistant to fibril formation; more prone to disaggregation, enzymatic digestion, and degradation; and hence less likely to cause neuronal damage and induce PD. In addition, AA interacts with syntaxin to regulate exocytosis of various neurotransmitters, including dopamine. AA has potent anti-inflammatory actions, serves as a mechanotransducer, influences cell membrane fluidity, enhances glucose uptake by neurons, possesses cytoprotective action, and is the precursor of anti-inflammatory lipoxin A4 (LXA4), actions that maintain the integrity of dopaminergic neurons in the substantia nigra pars compacta. The dynamic equilibrium among α-synuclein, syntaxin, AA, exocytosis of dopamine, formation of LXA4, and the inflammatory process is involved in the development of PD. This implies that deficiency of AA can render dopaminergic neurons susceptible to inflammatory neurodegeneration and development of PD. Hence, it is proposed that administration of AA and DHA (that are present in substantial amounts in the brain) can be used in the prevention and management of PD.

PubMed2027

AA in Brain-Memory, Dementia, Depression, Anxiety, Alzheimer's Disease, Affective and Psychiatric Disorders, and Coronary Heart Disease (CHD).

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

The human brain is rich in AA and DHA, which are crucial for normal brain growth and development and memory. They form a major structural component of neurons of the cerebral cortex and photoreceptor cells in the retina. AA and DHA support synaptic plasticity and contribute to efficient neurotransmission. Adequate amounts of AA and DHA support neuronal growth and the formation of new synapses and thus are crucial for memory and cognitive function.Depression, anxiety, schizophrenia, and other neurological diseases occur more frequently in those with coronary heart disease (CHD) and vice versa. Transient depression and anxious mood can trigger acute cardiac events and fatal arrhythmias. Serotonin and catecholamines play a significant role in depression and anxiety and are known to activate platelets, which increases the risk of cardiovascular events. Both serotonin and catecholamines modulate inflammation, while levels of pro-inflammatory cytokines are elevated in anxiety and depression and other psychiatric conditions and CHD. Depression, anxiety, and CHD involve altered essential fatty acid metabolism, particularly of AA and DHA, whose metabolites can inhibit platelet activation, suppress inflammation, and enhance acetylcholine levels. Acetylcholine, in turn, regulates the concentrations of serotonin and catecholamines. Acetylcholine and catecholamines have a modulatory influence on inflammation. Exercise is anti-inflammatory in nature, by virtue of its ability to enhance the formation of anti-inflammatory lipoxin A4, resolvins, protectin, and maresins and suppress pro-inflammatory cytokines IL-6 and TNF-α. AA and DHA enhance acetylcholine release and augment the formation of endothelial nitric oxide (NO). These results imply that anxiety, depression, and CHD are low-grade systemic inflammatory conditions that could benefit from the administration of appropriate amounts of AA and DHA. Risk factors for CHD such as obesity, type 2 diabetes mellitus, dyslipidemia, and atherosclerosis are all considered pro-inflammatory conditions.Since both neuropsychiatric conditions and recovery from CHD need the generation of new neurons and myocardial cells respectively that need to get integrated to the existing cells/tissues, it is proposed that a combination of brain-derived neurotrophic factor (BDNF) (that is needed for neuronal cells to survive), growth hormone (GH) (needed for growth of the newly generated neurons and myocardial cells), resolvins/lipoxins/protectins/maresins (needed to suppress inflammation and enhance wound healing), retinoic acid (needed for vertebrate development), and 1,4-DPCA that inhibits the excessive deposition of collagen and an inducer of formation of new blood vessels (angiogenesis) may be of significant benefit both in the prevention and management of neuropsychiatric conditions and CHD and their associated conditions such as obesity, diabetes mellitus, dyslipidemia, and atherosclerosis.

PubMed2027

AA in Hypertension and Preeclampsia.

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

Human essential hypertension is driven by a network of interacting nutritional, metabolic, inflammatory, and endothelial mechanisms in which AA and other PUFAs play a critical role. Beyond excess sodium intake, population data supports the role of inadequate calcium, potassium, and magnesium intake and low antioxidant vitamin status in shaping blood pressure regulation. These nutrients influence vascular smooth muscle tone and act as cofactors that support Δ6 and Δ5 desaturase activities, thereby governing tissue availability of AA, EPA, and DHA and downstream formation of vasodilator and antiplatelet mediators (e.g., PGE1, prostacyclin), as well as inflammation-resolving lipid mediators (lipoxins, resolvins, protectins, maresins, and nitrolipids).Endothelial dysfunction characterized by reduced endothelial nitric oxide (eNO) bioavailability and increased oxidative stress seems to play an important role in the pathobiology of HTN. High salt intake, asymmetric dimethylarginine (ADMA), and activation of NAD(P)H oxidase and angiotensin II signaling promote superoxide generation that quenches NO and shifts the vascular balance toward vasoconstriction. Clinical and experimental observations indicate that antihypertensive therapies can partly restore NO and antioxidant defenses, while dietary patterns rich in n-3 fatty acids (particularly DHA) and balanced n-6/n-3 intake may lower blood pressure by suppressing thromboxane formation and enhancing vasoprotective, pro-resolving pathways.Preeclampsia can be considered as a model of reversible hypertension linked to oxidative stress and angiogenic imbalance (sFlt1, soluble endoglin), highlighting mechanistic overlap with essential hypertension. Hypertension is a low-grade systemic inflammatory condition whose origins may be present in the perinatal period through long-term programming of PUFA metabolism and endothelial function.

PubMed2027

AA in Obesity and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Nonalcoholic Fatty Liver Disease (NAFLD).

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

Obesity and MASLD (NAFLD) are chronic, low-grade systemic inflammatory diseases resulting from complex interactions among diet, adipose tissue, gut microbiota, liver, muscle, pancreas, and the brain. Central to this integrative framework are AA and other polyunsaturated fatty acids (PUFAs), which regulate inflammation, insulin sensitivity, neuroendocrine signaling, and energy homeostasis.Excess consumption of energy-dense foods rich in saturated and trans fats leads to adipocyte hypertrophy, altered membrane fluidity, increased antigenicity, and enhanced recruitment of macrophages and T cells into adipose tissue. These changes promote the sustained release of pro-inflammatory cytokines TNF-α, IL-6, MIF, and CRP, contributing to insulin resistance, metabolic syndrome, and MASLD (NAFLD). In contrast, adequate availability of GLA, DGLA, AA, EPA, and DHA improves cell membrane dynamics and serves as a substrate for anti-inflammatory and pro-resolving lipid mediators, including lipoxins, resolvins, protectins, maresins, and nitrolipids, which suppress inflammatory signaling and enhance adiponectin production.Abdominal obesity can be considered a localized glucocorticoid excess state driven by increased adipose 11β-hydroxysteroid dehydrogenase-1 (11β-HSD-1) activity, linking visceral fat accumulation to insulin resistance, altered PPAR signaling, and chronic inflammation. Suppression of 11β-HSD-1, increased PUFA intake, and exercise can ameliorate these abnormalities.Gut microbiota and their short-chain fatty acids influence inflammation, leptin signaling, incretin secretion, and lipid metabolism, while PUFAs-including AA-activate gut-brain-liver neural circuits via vagal pathways and G protein-coupled receptors to regulate satiety, insulin secretion, and hepatic glucose production. This chapter also emphasizes the role of hypothalamic factors, particularly BDNF, in integrating nutritional, hormonal, and inflammatory signals.In summary, AA and related PUFAs are critical modulators of inflammation resolution, neuroendocrine control, and metabolic balance, offering a unifying mechanistic framework for the prevention and management of obesity and its associated metabolic disorders.

PubMed2027

AA in Rheumatoid Arthritis and Lupus.

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

Inflammation and immune dysregulation are at the center of various diseases, including autoimmune diseases, especially rheumatoid arthritis (RA) and systemic lupus erythematosus (lupus). In general, corticosteroids, disease-modifying drugs, and monoclonal antibodies against IL-6 and TNF-α, JAK inhibitors, and various other immunomodulators are extensively used in the treatment of RA and lupus. It is known that EFA (essential fatty acid) metabolism, especially that of AA, is altered in RA and lupus. It is proposed that metabolism of AA and of other PUFAs may play a role in predicting disease progression, prognosis, and response to treatments offered. For this discussion, a better understanding of the interaction between corticosteroids and EFA metabolism is needed.Corticosterone acts on three classes of PLA2 (calcium-independent PLA2 [iPLA2], secretory PLA2 [sPLA2], and cytosolic PLA2 [cPLA2]) and COX-2 and LOX to produce radical changes in the plasma and tissue concentrations of anti-inflammatory LXA4, resolvins, protectins, and maresins and pro-inflammatory (PGE2), leukotrienes (LTs), interleukin-1β (IL-1β), and platelet-activating factor (PAF) that modulate the inflammatory process. Studies revealed that corticosteroids inhibit the activities of Δ6 and Δ5 desaturases and, thus, decrease the formation of ω-6 (GLA, DGLA, AA) and ω-3 (EPA and DHA) that would limit the formation of both pro- and anti-inflammatory eicosanoids. Our previous studies revealed that AA and its metabolite LXA4 have potent anti-inflammatory effects. These results suggest that corticosteroids act at various stages of PUFA metabolism, implying that measurement of plasma levels of LA, ALA, GLA, DGLA, AA, EPA, DHA, LXA4, LTs, and TXs and cytokines may aid in predicting and monitoring the efficacy and potential adverse actions of corticosteroids and immunomodulators.

PubMed2027

AA-Induced Lipid Peroxidation at the Center of Tumor Cell Apoptosis and Ferroptosis.

Reviews of physiology, biochemistry and pharmacologyUndurti N Das

Polyunsaturated fatty acids (PUFAs), especially gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), can block HMG-CoA reductase activity and thus decrease cholesterol synthesis/formation. Cholesterol has antioxidant actions and thus reduces the formation of ROS (reactive oxygen species). In contrast, PUFAs augment the generation of ROS. PUFAs undergo peroxidation to form lipid peroxides that are toxic to tumor cells but not normal cells. GPX4 (glutathione peroxidase) reduces the formation of lipid peroxides and thus enhances tumor cell resistance to the tumoricidal actions of radiation, anticancer drugs, and immune checkpoint inhibitors (ICIs). Tumor cells have relatively high amounts of GPX4 and hence are resistant to lipid peroxide-induced ferroptosis/apoptosis. In contrast, normal cells can upregulate their GPX4 activity/content upon exposure to toxic lipid peroxides and hence are resistant to ferroptosis or apoptosis when supplemented with various PUFAs or exposed to radiation, anticancer drugs, and ICIs. Thus, PUFAs (especially GLA, AA, and DHA) are selectively toxic to tumors but not normal cells. Interferon-γ (IFN-γ) is secreted by CD8+ T cells, and AA induces ferroptosis of tumor cells in an ACSL4 (the protein encoded by this gene is an isozyme of the long-chain fatty-acid-coenzyme A ligase family)-dependent lipid peroxidation process. IL-6, TNF-α, and IFN-γ activate PLA2 to release AA from membrane lipids, which is then peroxidized to trigger apoptosis or ferroptosis of cancer cells. CD8+ T cells/macrophages/TILs/NK cells and other immunocytes downregulate the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system x c - , and impair the uptake of cystine by tumor cells, because of which accumulation of toxic lipid peroxides occurs in the tumor cells, leading to their apoptosis/ferroptosis/necrosis. In C6 glioma cells, the inhibition of the cystine/glutamate (XC) antiporter and glutamate-cysteine ligase (GCL) results in inhibition of glutathione biosynthesis that leads to ferroptosis of cancer cells because of accumulation of toxic lipid peroxides. Thus, the lipid peroxidation process is at the center of tumor cell apoptosis/ferroptosis.

PubMed2027

Advanced Image Generation for Cancer and Stem Cell Biology Using Diffusion Models.

Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder

Deep learning has transformed medical image analysis, but progress in cancer and stem cell applications is often constrained by limited access to large, diverse, well-annotated imaging datasets. This bottleneck is especially acute for studies of tumor heterogeneity and cancer stem cell (CSC) biology, where rare phenotypes and dynamic cell-state transitions-frequently linked to stemness-associated transcriptional programs (e.g., OCT4, SOX2, NANOG)-benefit from high-quality imaging across many samples and conditions. At the same time, regulatory and practical barriers (patient privacy, acquisition cost, and uneven institutional data sharing) restrict dataset scale and reuse. Diffusion models offer a practical route to synthetic data expansion by generating high-fidelity synthetic images that retain salient radiologic and pathologic features. In this chapter, we present an end-to-end protocol for adapting latent diffusion (Stable Diffusion) to oncology imaging using DreamBooth fine-tuning with small numbers of representative images, coupled with text-to-image and image-to-image workflows to generate controlled variations across modalities and disease presentations (e.g., brain tumor MRI, breast cancer mammography/CESM). We also describe quantitative and qualitative evaluation strategies, including Fréchet Inception Distance (FID) benchmarking and expert review considerations, to assess realism and diversity. These methods enable cancer and stem cell biologists to augment training data for segmentation and classification, build shareable educational resources, and prototype analyses for rare tumors or stemness-enriched subtypes while potentially reducing reliance on direct sharing of patient images.

PubMed2027

An Overview of Programmable Epigenetic Editing Based on CRISPR Tools for Gene Expression Regulation.

Methods in molecular biology (Clifton, N.J.)Marija Djordjevič, Svetlana Dinič, Ana Sarič, Aleksandra Uskokovič, Melita Vidakovič

Epigenetic regulation provides a dynamic and reversible layer of gene control that functions independently of changes in DNA sequence, primarily mediated by DNA methylation, histone modifications, and higher-order chromatin organization. Aberrant epigenetic states contribute to a wide range of human diseases. However, conventional epigenetic therapies based on small-molecule inhibitors lack locus specificity and often cause global chromatin disturbances. The emergence of programmable epigenetic editing technologies has transformed the field by enabling targeted rewriting of chromatin states at defined genomic loci. Catalytically inactive CRISPR/Cas9 platforms fused to transcriptional activators, repressors, or chromatin-modifying enzymes now allow precise addition or removal of epigenetic marks without altering the underlying DNA sequence. This chapter provides an overview of the conceptual and technical foundations of CRISPR-based epigenetic editing, including tools for gene activation and repression, DNA methylation, histone modifications, and multiplexed systems that permit coordinated regulation of multiple genomic loci or epigenetic marks. Delivery methods for in vitro and in vivo applications are discussed, with an emphasis on viral and nonviral platforms that enable tissue-specific, durable gene regulation. Finally, recent preclinical and clinical studies highlight the potential of programmable epigenetic editing as a next-generation therapy for precise and reversible gene control.

PubMed2027

Analysis of Cell Populations from Arbovirus-Infected Tissues by Flow Cytometry.

Methods in molecular biology (Clifton, N.J.)Jenniffer Ramos Martins, Viviane Lima Batista, Celso M Queiroz-Junior, Mauro Martins Teixeira, Vivian Vasconcelos Costa

Arboviruses still represent a major challenge to public health in many parts of the world. Despite shared epidemiological characteristics, each arboviral disorder displays distinct cellular tropism and activation patterns, progressing to immunopathological outcomes that remain not fully elucidated. A deeper understanding of these processes requires methodologies capable of characterizing the often-complex cellular responses. In this context, flow cytometry emerges as a valuable tool, enabling the characterization of heterogeneous cell populations, the assessment of activation and proliferation markers, the quantification of inflammatory mediators, and the detection of cell death pathways. By integrating these parameters, flow cytometry can enhance our understanding of arbovirus pathogenesis, clarify their cellular tropism, and understand immune responses to infection. This methodological article outlines the main steps for applying the technique to different target tissues infected by chikungunya, dengue, yellow fever, and Zika viruses, emphasizing the most effective strategies.

PubMed2027

Analysis of Chlamydia trachomatis Gene Function via Controlled Up- or Downregulation.

Methods in molecular biology (Clifton, N.J.)Colleen C Monahan, Nicole A Grieshaber, Scott S Grieshaber, Anders Omsland

Due to genome streamlining, many genes in the ~1 Mb genome of Chlamydia trachomatis are essential and cannot be deleted. Ectopic overexpression and CRISPRi-based knockdown are complementary genetic approaches to study the function of genes in C. trachomatis, including essential genes. When used in combination with inducible promoters, these tools allow researchers to determine how precise regulation of gene expression contributes to proper progression of the chlamydial developmental cycle. This chapter includes protocols which detail ectopic overexpression of a protein or conversely, transcriptional inhibition of a specific gene in C. trachomatis serovar L2/434/Bu.

PubMed2027

Analysis of Spatial Gene Expression in Chlamydia Using Fluorescent In Situ Hybridization.

Methods in molecular biology (Clifton, N.J.)Nicole A Grieshaber, Scott S Grieshaber

The obligate intracellular bacterium Chlamydia employs a unique, asynchronous biphasic developmental cycle characterized by distinct morphological forms: the replicative reticulate body (RB), the infectious elementary body (EB), and an intermediate body (IB). The simultaneous presence of these phenotypically distinct cell types throughout infection complicates the study of gene expression regulation during development. Conventional population-level assays are inadequate for dissecting the regulatory mechanisms within this mixed population. This chapter describes a robust method utilizing hybridization chain reaction (HCR) RNA fluorescence in situ hybridization (FISH) in conjunction with dual-promoter reporter Chlamydia strains to determine cell-form-specific transcript expression. We leverage dual-reporter strains to provide visual identification of RBs and EBs. Probes targeting genes of interest are designed to interact with spectrally distinct fluorescent HCR amplifiers. This versatile, multiplexing system allows for precise spatial and temporal localization of specific mRNAs within identified Chlamydial cell forms, offering a powerful tool to overcome current limitations in Chlamydia research and elucidate the regulatory underpinnings of its complex life cycle.

PubMed2027

Assessing the Vector Competence for Chikungunya Virus in Mosquitoes.

Methods in molecular biology (Clifton, N.J.)Alexander W E Franz

Chikungunya virus (CHIKV) is a human pathogenic, mosquito-borne virus (arbovirus), which is causing epidemic outbreaks among human populations in Africa, Asia, Europe, and South- and Central America including the Caribbean. The development of novel approaches to prevent mosquito transmission of the virus in the field requires a detailed study of CHIKV interactions with its mosquito vectors in the laboratory. In this chapter, we describe how to prepare Aedes aegypti mosquitoes for the infection with freshly cultivated CHIKV. We also describe quantitative and qualitative viral detection assays in mosquitoes based on plaque assays and the amplification of saliva samples in cell culture.

PubMed2027

Automated Counting of Chikungunya Virus-Formed Plaques.

Methods in molecular biology (Clifton, N.J.)Akino Emi, Youichi Suzuki

The cytopathic effect (CPE) is a key to understanding the pathogenicity of the chikungunya virus (CHIKV) and is useful for evaluating viral infectivity in vitro. The plaque assay, based on the CPE induced by viral infection, provides an infectious virus titer for test samples. Additionally, the assay is simple, quantitative, and cost-effective. Thus, the plaque assay remains the gold standard for determining the infectivity of CHIKV. However, manually counting of virus-induced plaques with the naked eye or under the microscope is often time-consuming and labor-intensive. Therefore, automated plaque-counting software would improve the consistency and efficiency of plaque counting. We recently developed plaQuest, a stand-alone Windows software that enables rapid, reliable plaque counting for CHIKV. In this chapter, we describe the basic procedure for detecting and counting the CHIKV-formed plaques in a 24-well plate.

PubMed2027

Bio-Inspired Silica Lipase Nanobiocatalysts for the Synthesis of Fatty Acid Methyl Esters.

Methods in molecular biology (Clifton, N.J.)Pablo Silva, Florencia Pirotti, Lorena Wilson, Lorena Betancor

Lipase immobilization strategies are key to the potential use of biological catalysts in reactions of interest. The bioinspired synthesis of silica nanoparticles uses biological or environmentally friendly processes to create highly functionalized supports with desirable properties such as increased surface area and stability. Here, we describe the preparation of two immobilization methods for Thermomyces lanuginosus lipase on biomimetic silica supports: in situ entrapment and covalent attachment to the hetero-functionalized surface of the nanoparticles, and their application in the synthesis of fatty acid methyl esters.

PubMed2027

Chikungunya Virus Infection in Paraffin-Embedded Tissue: Analysis of Histological Alterations and Viral Detection by Immunohistochemistry.

Methods in molecular biology (Clifton, N.J.)Natália G Salomão, Kíssila Rabelo

Histopathological analysis of tissues infected with Chikungunya virus (CHIKV), including biopsy and autopsy specimens, can provide valuable insights into the pathogenesis of atypical and fatal cases. By examining tissue architecture and cellular alterations under the microscope, it is possible to identify patterns of injury, inflammation, and cellular degeneration. These morphological findings provide evidence of how the pathogen interacts with host cells and tissues. Moreover, immunohistochemistry may be performed in tissue sections for the detection of viral antigens. Importantly, this technique allows the characterization of diverse biologically relevant targets in tissue samples, such as distinct immune cell subsets, cytokines, and additional molecular markers. This is accomplished by the use of antibodies selected for their specificity toward the antigen of interest. In this chapter, we present a concise overview of how histological analysis and immunohistochemical approaches can enhance the understanding of CHIKV-associated pathological mechanisms.

PubMed2027

Chlamydia trachomatis Infection in a Microphysiologic Cell Culture Model.

Methods in molecular biology (Clifton, N.J.)David J Delgado Diaz, Katherine M Nelson, Vonetta L Edwards, Ian J Glomski, Filipa Ribeiro, Patrik M Bavoil, Alison K Criss, Jacques Ravel,…

Chlamydia trachomatis is an obligate intracellular bacterium that infects the columnar epithelium of the human endocervix. While conventional two-dimensional cell cultures and animal models have been instrumental in advancing our understanding of C. trachomatis biology, they are limited in capturing the multicellularity, architecture, and physiological microenvironment of the human cervix. This chapter describes the use of a three-dimensional (3D) microphysiologic model to study Chlamydia trachomatis infection. The model is inexpensively made without specialized equipment and is designed to recreate the epithelial-stromal interface. We outline procedures for coculturing cervical epithelial cells and fibroblasts, infecting epithelial cells with fluorescently labeled C. trachomatis, monitoring infection progression via fluorescent microscopy, and quantifying infectious progeny. The complete developmental cycle of C. trachomatis within this model provides a robust and accessible platform to investigate C. trachomatis-specific host-pathogen interactions, immune responses, and the influence of diverse physiological and environmental stimuli within a relevant cervical context.

PubMed2027

Clinical Flow Cytometric Testing in Chronic Lymphocytic Leukemia.

Methods in molecular biology (Clifton, N.J.)Nadia Nasir, Maryalice Stetler-Stevenson, Hao-Wei Wang

Flow cytometry is the cornerstone for establishing the diagnosis of chronic lymphocytic leukemia (CLL), owing to its characteristic and well-defined immunophenotype that enables accurate distinction from other leukemias and lymphomas. Beyond diagnosis, flow cytometry provides essential prognostic information and allows sensitive detection of minimal residual disease (MRD), a strong predictor of clinical outcome. CLL MRD assessment is increasingly used to guide risk stratification, therapeutic decision-making, and treatment duration in the era of targeted therapies and immunotherapies. This chapter reviews best practices for specimen collection, processing, staining, and data analysis and summarizes the principles of flow cytometric MRD assessment in CLL.

PubMed2027

Collection and Processing of Adult Mosquitoes (Diptera: Culicidae) for Molecular Studies of Arbovirus Infection.

Methods in molecular biology (Clifton, N.J.)Griselda Ines Oria

The processing of adult mosquitoes collected in the field for studies of natural infection with arboviruses of public health interest (genera: Flavivirus, Alphavirus, Orthobunyavirus) follows the assembly of homogenized mosquito pools and extraction of genetic material using commercial or manual techniques, and the detection of viral RNA by polymerase chain reaction (nested RT-PCR) [11]. This paper describes the procedures for capturing, taxonomic determination, obtaining genetic material, and molecular techniques for detecting alphaviruses.

PubMed2027

Combined Lipidomic and Metabolomic Analyses on Cardiac Organoids.

Methods in molecular biology (Clifton, N.J.)Lorenzo Fontanelli, Carolina Ferri, Adrian Florentin Suman, Elisa Ceccherini, Giovanni Signore, Fabio Anastasio Recchia, Silvia Rocchiccioli

Cardiac organoids are increasingly used to model human cardiac development and disease, but their small size often limits molecular characterization, especially if different approaches and protocols are necessary to extract and quantify metabolites and lipids. Here, we present a multistep workflow for combined targeted free amino-acid (FAA) based metabolomic and lipidomic profiling from a single pooled cardiac organoid sample. The protocol covers organoid harvesting, detergent-assisted lysis, and a modified Folch extraction that generates an organic phase for lipid analysis and an aqueous phase for FAA metabolite analysis. Lipids are quantified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) in Multiple Reaction Monitoring (MRM) mode using class-matched external standards and an internal standard to support calibration and reduce technical variability. Free amino acids and derivatives are analyzed from the same sample after filtration, drying, and AccQ-Tag derivatization, with norvaline as an internal standard. Together, this approach maximizes information yield from limited material and enables integrated analysis of metabolic and lipid pathways within the same biological specimen, facilitating organoid-based studies of cardiac maturation, disease modeling, and pharmacological responses.

PubMed2027

Comprehensive Isolation Strategy for Intestinal Epithelial and Fibroblast Cells from Healthy and Tumor Human Tissues.

Methods in molecular biology (Clifton, N.J.)Samna Sagadevan, Paloma Ordóñez-Morán

Organoids and fibroblasts derived from patient tissue serve as physiologically meaningful in vitro models to investigate tissue biology, diseases, and treatment responses. In this study, we present a robust protocol for the simultaneous isolation and long-term culture of patient-derived organoids (PDOs) and fibroblasts from both healthy and colorectal cancer samples. The workflow uses enzymatic dissociation followed by efficient separation into epithelial and stromal cell fractions. Organoids are embedded in Matrigel or Matrigel-Collagen I mixtures to support three-dimensional growth, whereas fibroblasts are maintained on conventional two-dimensional culture dishes. This dual-culture approach facilitates a broad range of downstream applications.

PubMed2027

Computational Network Analysis for Defining Transcriptional Programs.

Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder

Cancer cell identity is governed by coordinated transcriptional programs that are frequently rewired during tumorigenesis. Systematic identification of cancer type-specific gene regulatory networks provides a framework for understanding oncogenic state transitions and for prioritizing candidate therapeutic targets. Here, we present a reproducible network-based workflow for reconstructing and analyzing transcriptional regulatory programs across human cancer types using publicly available expression datasets. We describe procedures for curating and preprocessing microarray data from the Gene Expression Omnibus, implementing random forest classification, and reconstructing gene regulatory networks using the CellNet platform. Detailed guidance is provided for evaluating classifier performance, quantifying network influence scores, integrating transcription factor, target interaction resources, and performing functional enrichment analyses. In addition, we outline approaches for comparing cancer-specific networks with corresponding normal tissue profiles to identify candidate drivers of malignant cell identity and potential prognostic biomarkers. Together, these protocols provide investigators with a scalable computational framework for defining cancer type-specific transcriptional states and for systematically interrogating regulatory mechanisms underlying tumor heterogeneity.

PubMed2027

Decoding Nano-Bio Interactions: Gene Regulatory Networks in Advanced Drug Loading.

Methods in molecular biology (Clifton, N.J.)Amin Alvani, Negar Mottaghi-Dastjerd, Abozar Ghorbani, Ahmad Gholami, Zeinab Pazhoohesh

Conventional treatments often face challenges such as the limited ability to penetrate the blood-brain barrier (BBB). The Doxorubicin-loaded graphene oxide/magnetite (DOX/GO/Fe3O4) nanocomplex offers a promising platform due to GO's high surface area and pH-sensitive release, and Fe3O4's magnetic properties. This protocol describes the methodology for evaluating the cytotoxicity of free DOX versus the DOX/GO/Fe3O4 nanocomplex in the A-172 glioblastoma cell line, followed by advanced bioinformatics analysis to identify gene networks and indirect pathways that enhance the nanomaterial's biocompatibility. The methodology integrates the MTT assay, real-time PCR for apoptosis genes (Casp3, Bax, and Bcl-2), and advanced analysis, including protein-protein interaction (PPI) networking, clustering, and promoter motif analysis. The analysis indicated that miR-92a-2-5p is a potential therapeutic target for preventing myocardial damage and enhancing biocompatibility. The findings highlight key regulatory pathways that indirectly boost nanodrug biocompatibility through the modulation of secondary components like miRNAs and cellular stress mechanisms.

PubMed2027

Determination of Chikungunya Virus Infectious Titer Using Plaque Assays.

Methods in molecular biology (Clifton, N.J.)Sebastiano Volpe, Claudia Filomatori

The plaque-forming assay is the gold-standard method for measuring infectious viral particles. In this technique, lytic viruses infect and destroy host cells, but their spread is restricted by a viscous overlay medium. As a result, new viral particles can only infect neighboring cells, leading to localized clear areas known as plaques, which become visible after staining the remaining living cells. The number of plaques reflects the number of infectious viral particles initially present in the sample and is reported as plaque-forming units (PFU) per sample volume. In this protocol, we describe step by step how to perform a plaque-forming assay to determine the concentration of chikungunya virus in a cell culture supernatant.

PubMed2027

Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Methods in molecular biology (Clifton, N.J.)Zhenlan Yao, Melody Li

Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.

PubMed2027

Experimental Evolution of Drug Resistance for Antibiotic Target Discovery in Chlamydia trachomatis.

Methods in molecular biology (Clifton, N.J.)Aakriti Singh, Barbara S Sixt

The recent application of experimental and computational drug discovery workflows has identified hundreds of new molecules that display potent, and in some cases highly selective, activity against bacteria of the genus Chlamydia. The full value of these novel antichlamydial molecules, however, depends critically on our ability to determine their molecular targets and modes of action. A particularly powerful and widely applied strategy for identifying candidate targets involves selecting for bacterial mutants that have acquired resistance to a compound's inhibitory activity, followed by determining the mutations responsible for this resistance. Such mutations often pinpoint the compound's direct target or reveal cellular pathways that modulate target engagement. Here, we present a detailed, step-by-step protocol for applying this approach to the clinically important, human-pathogenic species Chlamydia trachomatis. The workflow includes: (1) determining compound potency by quantitative dose-response analysis, (2) generating a resistant C. trachomatis mutant through serial passaging under progressively increasing selective pressure, (3) confirming the resistance phenotype, and (4) isolating bacterial genomic DNA for whole-genome sequencing to identify resistance-associated mutations and facilitate downstream target discovery.