In Vitro Reconstitution of Plant COPII Vesicles.

In vitro reconstitution studies enable the controllable and stepwise investigation of complicated biochemical processes. In yeast and mammals, in vitro reconstitution of COPII vesicles marked a pivotal point in characterizing the endoplasmic reticulum-to-Golgi anterograde trafficking route and revealed how vesicles mediate the selective and reliable transportation among topologically equivalent compartments. By providing the necessary physiological conditions in a cell-free environment, it enables the dissection of essential components required for the vesicle formation. To enrich and purify the small amount in vivo membrane-bounded compartments, it simplifies the evaluation of vesicle regulation by distinct external stimuli or upstream signals. Here, we describe the preparation of plant microsomes and cytosol for the reconstitution of plant COPII vesicles. Purified vesicles can be used for further biochemical or microscopical analyses.

Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.

Targeted degradation of membrane and extracellular proteins with LYTACs.

Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

The odyssey of the TR(i)P journey to the cellular membrane.

Ion channels are integral membrane proteins mediating ion flow in response to changes in their environment. Among the different types of ion channels reported to date, the super-family of TRP channels stands out since its members have been linked to many pathophysiological processes. The family comprises 6 subfamilies and 28 members in mammals, which are widely distributed throughout most tissues and organs and have an important role in several aspects of cellular physiology. It has been evidenced that abnormal expression, post-translational modifications, and channel trafficking are associated with several pathologies, such as cancer, cardiovascular disease, diabetes, and brain disorders, among others. In this review, we present an updated summary of the mechanisms involved in the subcellular trafficking of TRP channels, with a special emphasis on whether different post-translational modifications and naturally occurring mutagenesis affect both expression and trafficking. Additionally, we describe how such changes have been associated with the development and progress of diverse pathologies associated with the gain or loss of functional phenotypes. The study of these processes will not only contribute to a better understanding the role of TRP channels in the different tissues but will also present novel possible therapeutic targets in diseases where their activity is dysregulated.

Labor trafficking of migrant registered nurses.

BACKGROUND: Labor trafficking of registered nurses (RNs) in the USA impedes justice by denying inalienable human rights and equal economic opportunities. Nursing shortages in developed countries, poverty, social upheaval, and government actions influence migration, as do other factors related to determinants of health. Migrant RNs are visa workers, displaced, refugees, immigrants, or asylum seekers. Labor traffickers target vulnerable migrant RNs seeking employment outside their home country. Unlike ethical recruiters, traffickers lure migrant RNs into indentured contracts in work environments that result in health-threatening conditions, long shifts, and exorbitant fines that threaten families with financial retribution. PURPOSE: The purpose of the paper is to raise awareness. METHODS: Authors explain the background of influences and nuances in migrant RN labor trafficking. DISCUSSION: Identifying labor traffickers' deceitful, coercive, fraudulent, and illegal methods, assist organizational approaches for establishing Total Worker Health, trauma-informed care, coordinated community response, and No Door Closed actions when wanting to mitigate or eradicate labor trafficking of migrant RNs.

Using Interactive Television Instruction (ITV) to Reduce the Propensity to Engage in Substance Abuse and Drug Trafficking Among Vulnerable Adolescents in Nigeria.

This study examined the impact of ITV intervention on reduction in the propensity to abuse substances and engage in drug trafficking. The researcher conducted this study using an experiment of 517 vulnerable adolescents aged 10-19 years. The participants were randomly assigned to control (n = 258) and treatment (n = 259) groups. The researchers found a significant main effect of treatment conditions on reduction in the propensity to engage in substance abuse and drug trafficking among vulnerable adolescents. That is, before the intervention, there was no significant statistical difference between the control and treatment groups on the propensity to engage in substance abuse and drug trafficking. However, vulnerable children who received the intervention reported a significant reduction in propensity after the intervention. The results highlight the usefulness of ITV as a behaviour change strategy for vulnerable children.

Working With Youth Who Have Experienced Commercial Sexual Exploitation: Voices From the Field.

Little is known about the experiences of service providers working with youth who have experienced commercial sexual exploitation (CSE). In this qualitative study, 12 service providers shared their experiences working with youth who have experienced CSE. Thematic analysis was used to develop key themes that included the challenges posed by working with this population, including the breadth of necessary services and the type of support that is needed for this work. Participants shared the importance of being prepared to work with this population and learn from those with lived experience. The complexity of cases and the difficulty engaging youth who had experienced CSE were also themes. These results can inform service providers who work with youth about the common challenges in working with this population and prepare the next generation of service providers for this difficult work. Training service providers, managing their expectations, and getting them ready for their role will be critical for successful intervention with youth who have experienced CSE.

Visualization and Quantification of Organelle Axonal Transport in Cultured Neurons.

The specialized function and extreme geometry of neurons necessitates a unique reliance upon long-distance microtubule-based transport. Appropriate trafficking of axonal cargos by motor proteins is essential for establishing circuitry during development and continuing function throughout a lifespan. Visualizing and quantifying cargo movement provides valuable insight into how axonal organelles are replenished, recycled, and degraded during the dynamic dance of outgoing and incoming axonal traffic. Long-distance axonal trafficking is of particular importance as it encompasses a pathway commonly disrupted in developmental and degenerative disease states. Here, we describe neuronal organelles and outline methods for live imaging and quantifying their movement throughout the axon via transient expression of fluorescently labeled organelle markers. This resource provides recommendations for target proteins/domains and appropriate acquisition time scales for visualizing distinct neuronal cargos in cultured neurons derived from human induced pluripotent stem cells (iPSCs) and primary rat neurons.

Cellular mechanisms of acute rhabdomyolysis in inherited metabolic diseases.

Acute rhabdomyolysis (RM) constitutes a life-threatening emergency resulting from the (acute) breakdown of skeletal myofibers, characterized by a plasma creatine kinase (CK) level exceeding 1000 IU/L in response to a precipitating factor. Genetic predisposition, particularly inherited metabolic diseases, often underlie RM, contributing to recurrent episodes. Both sporadic and congenital forms of RM share common triggers. Considering the skeletal muscle's urgent need to rapidly adjust to environmental cues, sustaining sufficient energy levels and functional autophagy and mitophagy processes are vital for its preservation and response to stressors. Crucially, the composition of membrane lipids, along with lipid and calcium transport, and the availability of adenosine triphosphate (ATP), influence membrane biophysical properties, membrane curvature in skeletal muscle, calcium channel signaling regulation, and determine the characteristics of autophagic organelles. Consequently, a genetic defect involving ATP depletion, aberrant calcium release, abnormal lipid metabolism and/or lipid or calcium transport, and/or impaired anterograde trafficking may disrupt autophagy resulting in RM. The complex composition of lipid membranes also alters Toll-like receptor signaling and viral replication. In response, infections, recognized triggers of RM, stimulate increased levels of inflammatory cytokines, affecting skeletal muscle integrity, energy metabolism, and cellular trafficking, while elevated temperatures can reduce the activity of thermolabile enzymes. Overall, several mechanisms can account for RMs and may be associated in the same disease-causing RM.

SEC16A Variants Predispose to Chronic Pancreatitis by Impairing ER-to-Golgi Transport and Inducing ER Stress.

Chronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next-generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90% vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26-2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67-4.50) and in a biobank meta-analysis (OR, 1.16; 95% CI, 1.04-1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0 vs 45.0; p = 0.012). Functional studies using three CRISPR/Cas9-edited HEK293T cell lines show that loss-of-function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/- mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein-stimulated pancreatitis models, Sec16a+/- mice display heightened pancreatic inflammation and fibrosis compared to wild-type mice. These findings implicate a novel pathogenic mechanism predisposing to CP.

Targeting the MHC-I endosomal-lysosomal trafficking pathway in cancer: From mechanism to immunotherapy.

Immune checkpoint blockade (ICB) therapy has achieved broad applicability and durable clinical responses across cancer types. However, the overall response rate remains suboptimal because some patients do not respond or develop drug resistance. The low infiltration of CD8+ cytotoxic T cells (CTLs) in the tumor microenvironment due to insufficient antigen presentation is closely related to the innate resistance to ICB. The duration and spatial distribution of major histocompatibility complex class I (MHC-I) expression on the cell surface is critical for the efficient presentation of endogenous tumor antigens and subsequent recognition and clearance by CTLs. Tumor cells reduce the surface expression of MHC-I via multiple mechanisms to impair antigen presentation pathways and evade immunity and/or develop resistance to ICB therapy. As an increasing number of studies have focused on membrane MHC-I trafficking and degradation in tumor cells, which may impact the effectiveness of tumor immunotherapy. It is necessary to summarize the mechanism regulating membrane MHC-I translocation into the cytoplasm and degradation via the lysosome. We reviewed recent advances in the understanding of endosomal-lysosomal MHC-I transport and highlighted the means exploited by tumor cells to evade detection and clearance by CTLs. We also summarized new therapeutic strategies targeting these pathways to enhance classical ICB treatment and provide new avenues for optimizing cancer immunotherapy.

An acridone based fluorescent dye for lipid droplet tracking and cancer diagnosis.

Lipid droplets (LDs) are spherical organelles that localize in the cytosol of eukaryotic cells. Different proteins are embedded on the surface of LDs, so LDs play a vital role in the physiological activities of cells. The dysregulation of LDs is associated with various human diseases, such as diabetes and obesity. Therefore, it is essential to develop a fluorescent dye that labels LDs to detect and monitor illnesses. In this study, we developed the compound BDAA12C for staining LDs in cells. BDAA12C exhibits excellent LD specificity and low toxicity, enabling us to successfully stain and observe the fusion of LDs in A549 cancer cells. Furthermore, we also successfully distinguished A549 cancer cells and MRC-5 normal cells in a co-culture experiment and in normal and tumour tissues. Interestingly, we found different localizations of BDAA12C in well-fed and starved A549 cancer cells and consequently illustrated the transfer of fatty acids (FAs) from LDs to mitochondria to supply energy for ß-oxidation upon starvation. Therefore, BDAA12C is a promising LD-targeted probe for cancer diagnosis and tracking lipid trafficking within cells.

Understanding Child Sexual Exploitation Dynamics: Development and Validation of a Taxonomy of Recruitment and Domination Strategies.

Child sexual exploitation is a form of interpersonal violence which involves the use of manipulation, control, and coercion strategies to recruit and dominate minors. This study aimed to develop and validate a taxonomy that identifies, defines, and classifies these abusive strategies. The taxonomy was developed from an extensive literature review and its contents were validated via expert evaluation through a two-round Delphi method with 31 participants. Experts also judged the frequency of each strategy's usage to recruit and dominate the victims in sex trade, sex trafficking, sex tourism, and online sexual exploitation. The taxonomy comprises 20 specific strategies, classified into five categories: isolation, control of personal life, emotional abuse, cognitive manipulation, and behavioral domination. According to the experts, the strategy most frequently used to recruit the minors is "affective enticement", and the strategy that contributes most to the maintenance of the exploitative situation is "instrumentalization of sexuality". Nevertheless, different patterns were found regarding the frequency with which each strategy is used when considering the four manifestations of sexual exploitation separately. Delimiting the abusive strategies commonly perpetrated in child sexual exploitation improves our understanding of this complex phenomenon and could contribute to the development of effective prevention and intervention approaches.

Effect of size and pH-sensitivity of liposomes on cellular uptake pathways and pharmacokinetics of encapsulated gemcitabine.

To enhance cytoplasmic delivery efficiency, pH-sensitive liposomes (PSL) have been proposed as a novel strategy. To facilitate clinical translation, this study aims to understand the impact of both size and pH-sensitivity on cellular uptake pathways, intracellular trafficking and pharmacokinetics of liposomes. The large liposomes (130-160 nm) were prepared using thin-film hydration method, while small liposomes (∼60 nm) were fabricated using microfluidics, for both PSL and non-pH-sensitive liposomes (NPSL). Cellular uptake pathways and intracellular trafficking was investigated through confocal imaging with aid of various endocytosis inhibitors. Intracellular gemcitabine delivery by various liposomal formulations was quantified using HPLC, and the cytotoxicity was assessed via cell viability assays. Pharmacokinetics of gemcitabine loaded in various liposomes was evaluated in rats following intravenous administration. Larger liposomes had a higher loading capacity for hydrophilic gemcitabine (7% vs 4%). Small PSL exhibited superior cellular uptake compared to large PSL or NPSLs. Moreover, the alkalization of endosomes significantly attenuated the cellular uptake of PSL. Large liposomes (PSL and NPSL) predominantly entered cells via clathrin-dependent pathway, whereas small liposomes partially utilized caveolae-dependent pathway. However, the long circulation of the liposomes, as measured by the encapsulated gemcitabine, was compromised by both pH-sensitivity and size reduction (9.5 h vs 5.3 h). Despite this drawback, our results indicate that small PSL holds promise as vectors for the next generation of liposomal nanomedicine, owing to their superior cytoplasmic delivery efficiency.

Large liposomes had higher loading capacity for hydrophilic gemcitabine.Reduction of liposome size enhanced drug release from pH-sensitive liposomes.The internalization efficiency of liposomes was enhanced by pH-sensitivity and size reduction.Larger liposomes (>130 nm) enter cells primarily via clathrin-dependent endocytosis, while smaller liposomes (∼60 nm) partially through caveolae-mediated pathway, regardless of the pH-sensitivity.The intracellular payload release from pH-sensitive liposomes was decreased by endosome alkalization using chloroquine.Long circulation of the encapsulated gemcitabine was compromised by the pH-sensitivity and size reduction.

Loss of the putative Rab GTPase, Ypt7, impairs the virulence of Cryptococcus neoformans.

Small GTPases of the Rab family coordinate multiple membrane fusion and trafficking events in eukaryotes. In fungi, the Rab GTPase, Ypt7, plays a critical role in late endosomal trafficking, and is required for homotypic fusion events in vacuole biogenesis and inheritance. In this study, we identified a putative YPT7 homologue in Cryptococcus neoformans, a fungal pathogen causing life threatening meningoencephalitis in immunocompromised individuals. As part of an ongoing effort to understand mechanisms of iron acquisition in C. neoformans, we established a role for Ypt7 in growth on heme as the sole iron source. Deletion of YPT7 also caused abnormal vacuolar morphology, defective endocytic trafficking and autophagy, and mislocalization of Aph1, a secreted vacuolar acid phosphatase. Ypt7 localized to the vacuolar membrane and membrane contact sites between the vacuole and mitochondria (vCLAMPs), and loss of the protein impaired growth on inhibitors of the electron transport chain. Additionally, Ypt7 was required for robust growth at 39°C, a phenotype likely involving the calcineurin signaling pathway because ypt7 mutants displayed increased susceptibility to the calcineurin-specific inhibitors, FK506 and cyclosporin A; the mutants also had impaired growth in either limiting or high levels of calcium. Finally, Ypt7 was required for survival during interactions with macrophages, and ypt7 mutants were attenuated for virulence in a mouse inhalation model thus demonstrating the importance of membrane trafficking functions in cryptococcosis.

Unlocking cellular traffic jams: olive oil-mediated rescue of CNG mutant channels.

One of the reasons to suggest olive oil consumption for a healthy life is its potential to induce robust lipidomic remodeling through membrane modification by dietary lipids. This remodeling might, in turn, modulate essential lipid-protein interactions while maintaining accurate transmembrane protein/domain orientation. Oleic acid, the primary compound in olive oil, has been suggested as a modulator of ion channel function. In this study, we explored whether this lipid could rescue the trafficking of mutated transmembrane proteins. In our initial approach, we supplemented the cell culture medium of HEK-293 cells expressing cyclic nucleotide channels tagged using green fluorescent protein (CNG-GFP) with olive oil or oleic acid. In addition to wild-type channels, we also expressed R272Q and R278W mutant channels, two non-functional intracellularly retained channels related to retinopathies. We used fluorescence microscopy and patch-clamp in the inside-out configuration to assess changes in the cell localization and function of the tested channels. Our results demonstrated that olive oil and oleic acid facilitated the transport of cyclic nucleotide-gated R272Q mutant channels towards the plasma membrane, rendering them electrophysiologically functional. Thus, our findings reveal a novel property of olive oil as a membrane protein traffic inductor.

Fluorogenic RNA-based biomaterials for imaging and tracking the cargo of extracellular vesicles.

Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.

A review on dendrimer-based nanoconjugates and their intracellular trafficking in cancer photodynamic therapy.

Nanotechnology-based cancer treatment has received considerable attention, and these treatments generally use drug-loaded nanoparticles (NPs) to target and destroy cancer cells. Nanotechnology combined with photodynamic therapy (PDT) has demonstrated positive outcomes in cancer therapy. Combining nanotechnology and PDT is effective in targeting metastatic cancer cells. Nanotechnology can also increase the effectiveness of PDT by targeting cells at a molecular level. Dendrimer-based nanoconjugates (DBNs) are highly stable and biocompatible, making them suitable for drug delivery applications. Moreover, the hyperbranched structures in DBNs have the capacity to load hydrophobic compounds, such as photosensitizers (PSs) and chemotherapy drugs, and deliver them efficiently to tumour cells. This review primarily focuses on DBNs and their potential applications in cancer treatment. We discuss the chemical design, mechanism of action, and targeting efficiency of DBNs in tumour metastasis, intracellular trafficking in cancer treatment, and DBNs' biocompatibility, biodegradability and clearance properties. Overall, this study will provide the most recent insights into the application of DBNs and PDT in cancer therapy.

DBNs' intracellular journey in cancer-PDT refines targeted therapy, boosting efficacy.DBN in PDT for tumour metastasis: targeting and drug release mechanisms.DBNs' biocompatibility, biodegradability and clearance were explored thoroughly.

Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay.

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the ß-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The ß-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid's GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.