The role of γδ T cells in antitumor responses has gained significant attention due to their major histocompatibility complex (MHC)-independent killing mechanisms, which are functionally distinct from conventional αβ T cells. Notably, γδ tumor-infiltrating lymphocytes (TILs) have been identified as favorable prognostic markers in various cancers. However, the γδ TIL subsets, including Vδ1, Vδ2, and Vδ3, exhibit distinct prognostic implications and phenotypes within the tumor microenvironment (TME). Although the underlying mechanisms remain unclear, recent studies suggest that these subset-specific differences may arise from divergent activation pathways. Vδ1 TILs appear to be mainly activated by γδ T-cell receptor (TCR) signaling, whereas Vδ2 TILs seem to rely on alternative pathways, such as natural killer (NK) receptor-mediated activation. In addition to phenotypic studies, cancer immunotherapies, such as engineered γδ T cells, γδ T-cell engagers, and γδ TCR-based therapies, are under active development. However, despite these advancements, functional heterogeneity and limited persistence within TME remain significant challenges. Overcoming these obstacles could position γδ T-cell therapies as a transformative platform for cancer treatment. Here, we review recent findings on the prognostic significance of human γδ T cells, their phenotypic characteristics, and advances in γδ T-cell therapies, offering valuable insights for the development of novel cancer immunotherapies.

The laboratory mouse (Mus musculus) is the most widely used mammalian model organism in biomedical and life science research. This concise guide aims to provide essential information to assist researchers new to working with mice, covering topics such as mouse husbandry, maintenance, and available resources for obtaining mouse strains and associated data. Additionally, we discuss ethical considerations, emphasizing the 3Rs (replacement, reduction, and refinement) to ensure responsible and humane research practices.

Eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, which regulates all 3 unfolded protein response pathways, helps maintain cellular homeostasis and overcome endoplasmic reticulum (ER) stress through transcriptional and translational reprogramming. However, transcriptional regulation of mitochondrial homeostasis by eIF2α phosphorylation during ER stress is not fully understood. Here, we report that the eIF2α phosphorylation-activating transcription factor 4 (ATF4) axis is required for the expression of multiple transcription factors, including nuclear factor erythroid 2-related factor 2 and its target genes responsible for mitochondrial homeostasis during ER stress. eIF2α phosphorylation-deficient (A/A) cells displayed dysregulated mitochondrial dynamics and mitochondrial DNA replication, decreased expression of oxidative phosphorylation complex proteins, and impaired mitochondrial functions during ER stress. ATF4 overexpression suppressed impairment of mitochondrial homeostasis in A/A cells during ER stress by promoting the expression of downstream transcription factors and their target genes. Our findings underscore the importance of the eIF2α phosphorylation-ATF4 axis for maintaining mitochondrial homeostasis through transcriptional reprogramming during ER stress.

Recent advancements in fluorescence-based biosensor technologies have enabled more precise and accurate Förster Resonance Energy Transfer (FRET) imaging within Agrobacterium-mediated plant transformation systems. However, the application of FRET imaging in plant tissues remains hindered by significant challenges, particularly the time-intensive process of generating transgenic lines and the complications arising from tissue autofluorescence. In contrast, protoplast-based FRET imaging offers a rapid and efficient platform for functional screening and analysis, making it an essential tool for plant research. Nevertheless, conventional protoplast-based FRET approaches are often limited by background interference, inconsistent imaging conditions, and difficulties in quantitative analysis. Here, we present a systematic optimization of imaging conditions using the calcium biosensor D3cpv, addressing these limitations to improve both precision and efficiency in protoplast-based FRET imaging. This work serves as a practical guide for streamlining FRET imaging workflows and maximizing the utility of biosensors in plant cell studies.

Ralstonia solanacearum causes bacterial wilt, a devastating disease in solanaceous crops. The pathogenicity of R. solanacearum depends on its type III secretion system, which delivers a suite of type III effectors into plant cells. The disordered core effector RipAO is conserved across R. solanacearum species and affects plant immune responses when transiently expressed in Nicotiana benthamiana. Specifically, RipAO impairs pathogen-associated molecular pattern-triggered reactive oxygen species production, an essential plant defense mechanism. RipAO fused to yellow fluorescent protein initially localizes to filamentous structures, resembling the cytoskeleton, before forming large punctate aggregates around the nucleus. Consistent with these findings, tubulin alpha 6 (TUA6) and tubulin beta-1, building blocks of microtubules, were identified as putative targets of RipAO in immunoprecipitation and mass spectrometry analyses. In the presence of RipAO, TUA6-labeled microtubules fragmented into puncta, mimicking the effects of oryzalin, a microtubule polymerization inhibitor. These effects were corroborated in a N. benthamiana transgenic line constitutively expressing green fluorescent protein-labeled TUA6, where RipAO reduced microtubule density and stability at an accumulation level that did not induce aggregation. Moreover, oryzalin treatment further enhanced RipAO's impairment of reactive oxygen species production, suggesting that RipAO disrupts microtubule networks via its association with tubulins, leading to immune suppression. Further research into RipAO's interaction with the microtubule network will enhance our understanding of bacterial strategies to subvert plant immunity.

Immunostaining is an essential biological technique that determines the localization and level of target antigen molecules using antibodies within cells or tissues. Here, we present a brief guide to immunostaining, including the principles, methods, and different types of immunostaining. This manuscript will also provide common challenges and optimization strategies. This work will be useful for researchers with basic knowledge in immunostaining.

The clinical manifestations of Parkinson's disease (PD) are driven by aggregation of α-Synuclein (α-Syn) in the brain. However, there is increasing evidence that PD may be initiated in the gut and thence spread to the brain, eg, via the vagus nerve. Many studies link PD to changes in the gut microbiome, and bacterial amyloid has been shown to stimulate α-Syn aggregation. Yet, we are not aware of any studies reporting on a direct connection between microbiome components and α-Syn aggregation. Here, we report that soluble extract from the gut microbiome of the rats, particularly young rats transgenic for PD, shows a remarkably strong ability to inhibit in vitro α-Syn aggregation and keep it natively unfolded and monomeric. The active component(s) are heat-labile molecule(s) of around 30- to 100-kDa size, which are neither nucleic acid nor lipid. Proteomic analysis identified several proteins whose concentrations in different rat samples correlated with the samples' anti-inhibitory activity, while a subsequent pull-down assay linked the protein chaperone DnaK with the inhibitory activity of young rat's microbiome, confirmed in subsequent in vitro assays. Remarkably, the microbiome extracts also protected neuroblastoma SH-SY5Y cells and zebrafish embryos against α-Syn toxicity. Our study sheds new light on the gut microbiome as a potential source of protection against PD and opens up for new microbiome-based therapeutic strategies.

The base excision repair (BER) pathway is a critical mechanism in genomic stability. This review investigates the role of the BER pathway in advanced cancer therapies considering the pivotal role of genetic factors in cancer patient responses and prognosis. BER factors significantly influence genetic instability and cancer prognosis, as well as the effectiveness of chemotherapy and radiation therapy. In various cancers such as breast, colon, lung, and bladder, BER factors have shown potential as critical biological markers for predicting cancer outcomes. This study focuses on the polymorphisms and expression levels of key BER genes, including OGG1, XRCC1, APE1, and Polβ. Our findings demonstrate that the expression levels of BER genes and proteins are closely associated with the risk, progression, treatment response, and prognosis of various cancers. These insights could improve cancer treatments and aid in the development of drugs targeting BER proteins. Ongoing research in this field requires extensive statistical analyses and large-scale prospective studies to effectively utilize BER protein levels. Ultimately, these results suggest that the BER pathway represents a potential target for cancer diagnosis, prognostic prediction, and the development of personalized therapeutic strategies. This paves the way for effective cancer treatment in the future.

It is well-established that large and heavy dogs tend to live shorter lives. In this study, we aimed to determine whether traits other than body size are associated with the life expectancy of dogs. We compiled a dataset of 20 phenotypes, including body size, lifespan, snout ratio, and shedding, into a single matrix for 149 dog breeds using data from the American Kennel Club and other peer-reviewed sources. The analysis revealed that drooling might be associated with both the lifespan and body mass index of dogs. Furthermore, a genome-wide association study with adjusted phenotypes and statistical verification methods, such as Mendelian randomization. Additionally, conducting differential gene expression analysis with the salivary gland for the 2 cases, hypersalivation/less drooling vs various body sizes, we could observe the hypersalivation-related proteins. This genetic analysis suggests that body size and drooling might be candidate factors influencing lifespan. Consequently, we identified several candidate genes, including IGSF1, PACSIN2, PIK3R1, and MCCC2, as potential genetic factors influencing longevity-related phenotypes.

Despite the fact that endometriosis is a common gynecological disease that occurs in 10% of women of reproductive age, the pathogenesis and treatment strategy are not clear to date. Endometriosis patients are commonly characterized by adhesions in the pelvis or ovaries, which leads to prolonged inflammation in the abdominal cavity. To handle the chronic inflammation, changes of immune cells, including T cells, NK cells, and macrophage, are accompanied. Therefore, diverse cytokines and adhesions of the abdominal cavity lead to poor quality of ovarian follicles, inappropriate response to the hormone, and infertility. This review will guide researchers to summarize the molecular changes and identify new treatment strategies for endometriosis-mediated inflammation and pregnancy failure.

Amyloid precursor protein (APP) is a single-pass transmembrane protein abundantly expressed in the central nervous system and implicated in familial Alzheimer's disease, a progressive neurodegenerative disorder that impairs memory. Here, we investigated the role of amyloid precursor protein-like (APPL) using the model organism Drosophila melanogaster. In this study, Appl null mutants exhibited a reduced lifespan under normal conditions and increased triglyceride levels, which were mitigated by metformin treatment. Additionally, taste-associative memory impairment in Appl mutants suggested APPL's role in memory formation, which was restored by curcumin supplementation. The Appl mutants also displayed reduced climbing ability, which was improved by supplementation with vitamins C (ascorbic acid) and B (riboflavin). These findings suggest that APPL is involved in metabolic regulation, cognition, climbing activity, and aging in Drosophila melanogaster.

Microrchidia family CW-type zinc finger 2 (MORC2) is a nuclear protein that has been highly conserved throughout evolution. MORC2 consists of an ATPase domain at the N-terminus, a CW-type zinc finger domain in the middle, and coiled-coil domains at the C-terminus. MORC2 is involved in various important biological processes such as transcriptional regulation, chromatin remodeling, DNA damage repair, and metabolism. Recent studies suggest that MORC2 may serve as a potential biomarker and therapeutic target for hereditary neurological diseases and cancers. However, the exact molecular functions and pathogenic mechanisms of MORC2 in human diseases remain to be explored. In this review, we provide an overview of recent advancements in understanding the molecular functions of MORC2, as well as the characteristics and mechanisms of MORC2-related diseases, which will be valuable for future studies.

The polyextremophilic bacterium Aeribacillus pallidus produces a thermo- and alkali-stable γ-carbonic anhydrase (γ-apCA), a homotrimeric metalloenzyme containing a zinc ion in its active site that catalyzes the reversible hydration of carbon dioxide (CO). Here, we present the first crystal structure of γ-apCA at 1.7-Å resolution, revealing 2 trimers in the asymmetric unit. The overall structure is consistent with other γ-CAs, where each monomer adopts a prism-like structure consisting of an N-terminal left-handed β-helix and a C-terminal α-helix. The active site, located at the interface between 2 monomers, coordinates the zinc ion with 3 histidine residues (H65, H82, and H87) and a water molecule in a tetrahedral configuration. The structural comparison indicates that the amino acid composition at the active site of γ-apCA differs significantly from the prototypic γ-CA from Methanosarcina thermophila. This variation likely accounts for the lack of measurable CO hydration activity in γ-apCA. Additionally, the structure reveals noncatalytic zinc and sulfate ions trapped at the trimer core and trimer-trimer noncrystallographic interfaces. These may contribute to stabilizing enzyme assembly and promoting crystal packing.

Transcription is an essential biological process involving numerous factors, including transcription factors (TFs) which play a central role in this process by binding to their cognate DNA motifs. Although cells must tightly regulate the kinetics of factor association and dissociation during transcription, factor dynamics during transcription remain poorly characterized, primarily because of the reliance on ensemble experiments that average out molecular heterogeneity. Recent advances in single-molecule fluorescence imaging techniques have enabled the exploration of TF dynamics at unprecedented resolution. Findings on the temporal dynamics of individual TFs have challenged classical models and provided new insights into transcriptional regulation. Single-molecule imaging has also elucidated the assembly kinetics of transcription complexes. In this review, we describe the single-molecule fluorescence imaging methods widely used to determine factor dynamics during transcription. We highlight new findings on TF binding to chromatin, TF target search, and the assembly order of transcription complexes. Additionally, we discuss the remaining challenges in achieving a comprehensive understanding of the temporal regulation of transcription.

The long pentraxin 3 (PTX3), a marker of inflammation, has been associated with cardiovascular disease, obesity, and metabolic syndrome. Recently, elevated serum PTX3 levels have been linked to type 2 diabetes in obese patients with nonalcoholic fatty liver disease. Diabetes mellitus is a metabolic syndrome characterized by hyperglycemia resulting from insufficient insulin secretion or action. However, the precise role of PTX3 in hyperglycemia remains unclear. This study aimed to investigate the physiological roles of PTX3 in vivo. The deformation of pancreatic islets was mitigated in PTX3-deficient mice treated with streptozotocin (STZ) compared to control C57BL/6J mice. In addition, PTX3 deficiency prevented STZ-induced unfolded protein responses and pancreatic β-cell death. Immunoblotting data revealed significant inhibition of inositol-requiring protein1α and C/EBP homologous protein (CHOP) protein expression in PTX3 KO mice administered tunicamycin which is a chemical endoplasmic reticulum stress inducer. Similarly, tunicamycin-induced Grp78, Grp94, ATF6, and CHOP mRNA levels were reduced in PTX3 KO mice. Moreover, recombinant PTX3-induced CHOP expression and β-cell apoptosis in primary mouse islets. These findings suggest that PTX3 plays a critical role in STZ-induced deformation of pancreatic islets via regulating endoplasmic reticulum stress and β-cell apoptosis.

Dipeptidyl peptidase 4 (DPP4) is a membrane-bound protease known for its roles in immunity and metabolism; however, its function in the nervous system remains largely unexplored. We found that DPP4 is predominantly expressed in the Schwann cells of the sciatic nerve, and its systemic depletion in postnatal mice resulted in a decline in motor function. Importantly, the inhibition of its proteolytic activity did not affect axon regeneration, indicating that DPP4's protease activity may not be directly involved in axon regeneration. Instead, we observed a reduction in DPP4 protein levels in the sciatic nerve after injury and increased in serum postinjury, suggesting that DPP4 may be shed into circulation, potentially mediating systemic responses following injury. These findings highlight DPP4's importance in sensory function and its potential role in systemic responses after peripheral nerve injury.

Phytophthora species, an oomycete plant pathogen, secrete effectors into plant cells throughout their life cycle for manipulating host immunity to achieve successful colonization. However, the molecular mechanisms underlying effector-triggered necrotic cell death remain elusive. In this study, we identified an RXLR (amino acid residue; Arginine-Any amino acid-Leucine-Arginine motif) effector (Pc12) from Phytophthora capsici, which contributes to virulence and induces necrosis by triggering a distinct endoplasmic reticulum (ER) stress response through its interaction with Rab13-2. The necrotic cell death induced by Pc12 did not exhibit conventional effector-triggered immunity-mediated hypersensitive cell death, including the involvement of nucleotide-binding site leucine-rich repeat downstream signaling components and transcriptional reprogramming of defense-related genes. Instead, it alters the localization of ER-resident proteins and confines secretory proteins within the ER. Pc12 directly interacts with Rab13-2, which is primarily localized to the ER and Golgi apparatus, resulting in a diminished Rab13-2 signal on the Golgi apparatus. Furthermore, Rab13-2 exhibits increased affinity for its interactor, Rab escort protein 1, in the presence of Pc12. Structural predictions revealed that a specific residue of Rab13-2 is crucial for binding to the C-terminus of Pc12. Substitution of this residue reduced its interaction with Pc12 and impaired P. capsici infection while maintaining its interaction with Rab escort protein 1 and prenylated Rab acceptor 1. These findings provide insight into how a pathogen effector induces a distinct form of necrotic cell death to facilitate colonization of the host plant by disrupting the recycling of Rab13-2, a protein involved in vesicle trafficking at the ER-Golgi interface.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor impairments and the accumulation of misfolded α-synuclein. Dysregulation of the autophagy-lysosomal pathway (ALP), responsible for degrading misfolded proteins, has been implicated in PD pathogenesis. However, current diagnostic approaches rely heavily on motor symptoms, which occur due to substantial neurodegeneration, limiting early detection and intervention. This study investigated the potential of ALP-associated proteins in peripheral blood mononuclear cells (PBMCs) as diagnostic biomarkers for early-stage PD. Quantitative analysis revealed a significant reduction in optineurin levels in PBMCs from PD patients, and the expression levels of various ALP-associated proteins were tightly correlated, suggesting a coordinated dysregulation of the pathway. Correlation analyses revealed associations between ALP-associated features and clinical characteristics, such as age of onset and motor impairment. Furthermore, the study identified multiple positive correlations among ALP-associated proteins and functional readouts, highlighting the interconnectivity within the pathway. Notably, a PBMC biomarker model incorporating lysosomal-associated membrane protein 1 and optineurin exhibited high diagnostic accuracy (86%) in distinguishing PD patients from controls. These findings highlight the potential of ALP-associated protein signatures in PBMCs as novel diagnostic biomarkers for early detection and intervention in PD, offering insights into the systemic manifestations of the disease.

Plasma membrane proteins are crucial for signal transduction, trafficking, and cell-cell interactions, all of which are vital for cell survival. These proteins, including G-protein coupled receptors, ion channels, transporters, and receptors, are key drug targets due to their central role in receiving and amplifying cellular signals. However, the isolation and purification of plasma membrane proteins pose significant challenges because of their integration with phospholipid bilayers and the small fraction of these proteins present in the plasma membrane. Biotinylation, in combination with streptavidin beads, provides an effective method for surface protein analysis by specifically labeling surface proteins without penetrating the cell membrane, enabling precise isolation and analysis with minimal contamination. In this study, we describe a 1-step method for analyzing plasma membrane proteins that can be routinely implemented in many laboratories.

Various approaches employing cytokines and cytokine gene-modified tumor cells have been explored to induce antitumor responses, yet their widespread application has been limited due to efficacy concerns and adverse effects. In this study, interleukin-7 was engineered for expression both as a natural secretory form (sIL-7) and as a membrane-bound form fused with the B7.1 type I transmembrane protein (mbIL-7/B7) on CT26 colon cancer cells. Analysis of the resulting cell clones demonstrated that ectopically expressed sIL-7 and mbIL-7/B7 both retained similar capacities to induce the expansion and activation of CD8 T cells and to enhance antitumor responses in vitro. While the sIL-7 or mbIL-7/B7 clones showed similar growth in culture, the mbIL-7/B7 clone exhibited lower tumorigenicity in mice compared with the sIL-7 clone or wild-type CT26 cells. Specifically, the mbIL-7/B7 clone failed to form tumors in approximately 60% of the mice injected with it. Moreover, 80% of mice that rejected the mbIL-7/B7 clone developed long-term systemic immunity against CT26 cells. Analysis of immune cells within the tumor masses revealed significant increases in CD4 T cells, CD8 T cells, and dendritic cells in tumors formed by the mbIL-7/B7 clone compared to those formed by the sIL-7 clone. These findings suggest that the membrane-bound form of IL-7 with B7.1 is more effective than the secretory form in establishing antitumor immunity within the tumor microenvironment. Our strategy of expressing the mbIL-7/B7 chimera holds promise as a novel approach for tumor therapy, particularly in cases requiring IL-7 supplementation.