Waste paper-derived porous carbon via microwave-assisted activation for energy storage and water purification.

The reuse of waste papers by conversion into valuable carbon materials has received considerable attention for diverse applications such as energy storage and water purification. However, traditional methods for converting waste papers into materials with suitable properties for specific applications are often complex and ineffective, involving consecutive carbonization and activation steps. Herein, we propose a simple one-step microwave (MW)-assisted synthesis for preparing waste paper-derived porous carbons (WPCs) for energy storage and water purification. Through a 30-min synthesis, WPCs with graphitic structure and high specific surface area were successfully produced. The fabricated WPCs exhibited outstanding charge storage capability with a maximum specific capacitance of 237.7 F g-1. Additionally, the WPC demonstrates a high removal efficiency for various dyes, achieving a maximum removal efficiency of 95.0% for methylene blue. The developed one-step MW synthesis not only enables the production of porous carbon from waste paper, but also offers a viable approach to address solid waste management challenges while simultaneously yielding valuable materials.

A comprehensive Exdia TRF-LFIA for simultaneous quantification of GFAP and NT-proBNP in distinguishing ischemic and hemorrhagic stroke.

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Evaluation of the anti-cancer efficacy of lipid nanoparticles containing siRNA against HPV16 E6/E7 combined with cisplatin in a xenograft model of cervical cancer.

OBJECTIVE: To investigate the anti-cancer efficacy of ENB101-LNP, an ionizable lipid nanoparticles (LNPs) encapsulating siRNA against E6/E7 of HPV 16, in combination therapy with cisplatin in cervical cancer in vitro and in vivo. METHODS: CaSki cells were treated with ENB101-LNP, cisplatin, or combination. Cell viability assessed the cytotoxicity of the treatment. HPV16 E6/E7 gene knockdown was verified with RT-PCR both in vitro and in vivo. HLA class I and PD-L1 were checked by flow cytometry. A xenograft model was made using CaSki cells in BALB/c nude mice. To evaluate anticancer efficacy, mice were grouped. ENB101-LNP was given three times weekly for 3 weeks intravenously, and cisplatin was given once weekly intraperitoneally. Tumor growth was monitored. On day 25, mice were euthanized; tumors were collected, weighed, and imaged. Tumor samples were analyzed through histopathology, immunostaining, and western blot. RESULTS: ENB101-LNP and cisplatin synergistically inhibit CaSki cell growth. The combination reduces HPV 16 E6/E7 mRNA and boosts p21 mRNA, p53, p21, and HLA class I proteins. In mice, the treatment significantly blocked tumor growth and promoted apoptosis. Tumor inhibition rates were 29.7% (1 mpk ENB101-LNP), 29.6% (3 mpk), 34.0% (cisplatin), 47.0% (1 mpk ENB101-LNP-cisplatin), and 68.8% (3 mpk ENB101-LNP-cisplatin). RT-PCR confirmed up to 80% knockdown of HPV16 E6/E7 in the ENB101-LNP groups. Immunohistochemistry revealed increased p53, p21, and HLA-A expression with ENB101-LNP treatments, alone or combined. CONCLUSION: The combination of ENB101-LNP, which inhibits E6/E7 of HPV 16, with cisplatin, demonstrated significant anticancer activity in the xenograft mouse model of cervical cancer.

A novel small molecule, CU05-1189, targeting the pleckstrin homology domain of PDK1 suppresses VEGF-mediated angiogenesis and tumor growth by blocking the Akt signaling pathway.

Inhibition of angiogenesis is considered a promising therapeutic approach for cancer treatment. Our previous genetic research showed that the use of a cell-penetrating peptide to inhibit the pleckstrin homology (PH) domain of 3-phosphoinositide-dependent kinase 1 (PDK1) was a viable approach to suppress pathological angiogenesis. Herein, we synthesized and characterized a novel small molecule, CU05-1189, based on our prior study and present evidence for the first time that this compound possesses antiangiogenic properties both in vitro and in vivo. The computational analysis showed that CU05-1189 can interact with the PH domain of PDK1, and it significantly inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells without apparent toxicity. Western blot analysis revealed that the Akt signaling pathway was specifically inhibited by CU05-1189 upon VEGF stimulation, without affecting other VEGF receptor 2 downstream molecules or cytosolic substrates of PDK1, by preventing translocation of PDK1 to the plasma membrane. We also found that CU05-1189 suppressed VEGF-mediated vascular network formation in a Matrigel plug assay. More importantly, CU05-1189 had a good pharmacokinetic profile with a bioavailability of 68%. These results led to the oral administration of CU05-1189, which resulted in reduced tumor microvessel density and growth in a xenograft mouse model. Taken together, our data suggest that CU05-1189 may have great potential and be a promising lead as a novel antiangiogenic agent for cancer treatment.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Plasma Glial Fibrillary Acidic Protein and N-Terminal Pro B-Type Natriuretic Peptide: Potential Biomarkers to Differentiate Ischemic and Hemorrhagic Stroke.

Acute stroke management is critically time-sensitive and challenging. Blood-based biomarkers that can differentiate acute ischemic stroke (IS) from hemorrhagic stroke (HS) can greatly facilitate triage and early management. Admission blood samples obtained within 6 h of stroke symptom onset were analyzed in a derivation/validation design. GFAP, N-FL, NT-proBNP, copeptin, neutrophils (%), NLR, and platelet counts were assessed in the derivation cohort. The informative markers and the derived cutoff values were evaluated in the validation cohort. GFAP > 703 pg/mL showed a PPV of 76.9% and NPV of 95.8% for differentiating HS from IS. Multiple logistic regression analysis showed that GFAP and NT-proBNP were independent variables associated with IS and HS differentiation. Furthermore, applying a combined cutoff (GFAP > 703 pg/mL and NT-proBNP ≤ 125 pg/mL) for HS detection increased the PPV in both the derivation and validation cohorts (93.3% and 100%, respectively). GFAP and NT-proBNP levels were validated as informative blood biomarkers in the differentiation of IS and HS and using a combination of GFAP and NT-proBNP is suggested as a feasible strategy to differentiate stroke subtypes in the hyperacute phase of stroke.

Fabrication of Versatile Janus Microparticles through Geometry and Surface Chemistry Control.

Amphiphilic Janus particles typically comprise two distinct hemispheres with spatially dispersed physicochemical properties. The anisotropic structure and physicochemical properties of Janus particles can be exploited for various applications. However, their preparation typically requires complex and sophisticated processes and expensive equipment to control the formation of different structures and chemical compositions. Herein, a simple synthetic approach for the facile fabrication of versatile Janus particles with efficient control of the Janus ratio and wettability based on particle fixation at a three-phase interface and photopolymerization is reported. Agarose gel and surfactant are used to control the surface-coated boundaries of the Janus particles through the equilibrium of a floating microparticle at the fluid interface. poly(propylene glycol) diacrylate or poly(N-isopropylacrylamide) coating on polystyrene-based microparticles allows easy control of the chemical functionality of the particle surfaces. Depending on the particle morphology and wettability, the interfacial behavior between two immiscible liquids can be adjusted, which allows the stabilization of Pickering emulsions that encapsulate independent oil droplets in water or vice versa. This facile approach has the potential to enable more efficient mass production of Janus particles and their use in various applications, such as biomedical and environmental engineering.

CU06-1004 as a promising strategy to improve anti-cancer drug efficacy by preventing vascular leaky syndrome.

Background: Interleukin-2 (IL-2) is the first cancer therapeutic agent with an immunomodulatory function. Although it has been experimentally proven to be effective against metastatic renal cell carcinoma and metastatic melanoma, the clinical application of high-dose IL-2 (HDIL-2) has been limited because of its short half-life and severe side effects, such as vascular leakage syndrome (VLS) or capillary leaky syndrome (CLS). However, methods for overcoming this issue have not yet been identified. Methods: We discovered CU06-1004, an endothelial dysfunction blocker, through a previous study, and co-treated with IL-2 immunotherapy to confirm its inhibitory effect on HDIL-2-induced endothelial permeability. CU06-1004 was co-administered with HDIL-2 for 4 days in an in vivo mouse model. After drug injection, the mice were sacrificed, and Evans blue staining was performed. Results: In vitro, HDIL-2 treatment decreased HUVEC stability, which was rescued by co-treatment with CU06-1004. In our mouse model, co-administration of CU06-1004 and HDIL-2 prevented HDIL-2-induced vascular leakage by normalizing endothelial cells. Notably, the HDIL-2 and CU06-1004 combination therapy considerably reduced tumor growth in the B16F10 melanoma mouse model. Conclusion: Our data suggest that CU06-1004 acts as a potential anticancer drug candidate, not only by preventing HDIL-2-induced VLS but also by enhancing the anticancer effects of HDIL-2 immunotherapy.

Efficacy of CU06-1004 via regulation of inflammation and endothelial permeability in LPS-induced acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening condition that fundamentally results from inflammation and edema in the lung. There are no effective treatments available for clinical use. Previously, we found that as a leakage blocker CU06-1004 prevents endothelial barrier disruption and enhances endothelial cell survival under inflammatory conditions. In this study, we aimed to elucidate the effect of CU06-1004 in terms of prevention of inflammation and endothelial dysfunction in an ALI mouse model. METHODS: An ALI model was established that included intraperitoneal administration of LPS. Following LPS administration, survival rates and lung wet/dry ratios were assessed. Histological analysis was performed using hematoxylin and eosin staining. Scanning electron microscopy was used to examine alveolar and capillary morphology. Cytokines such as IL-1ß, IL-6, and TNF-α were analyzed using an ELISA assay of bronchoalveolar lavage fluid (BALF) and serum. Neutrophil infiltration was observed in BALF using Wright-Giemsa staining, and myeloperoxidase (MPO) activity was assessed. Pulmonary vascular leakage was confirmed using Evans-blue dye, and the expression of junctional proteins was evaluated using immunofluorescent staining. Expression of adhesion molecules was observed using immunofluorescence staining. NF-κB activation was determined using immunohistochemistry and western blot analysis. RESULTS: Survival rates and pulmonary edema were ameliorated with CU06-1004 treatment. Administration of CU06-1004 normalized histopathological changes induced by LPS, and alveolar-capillary wall thickening was reduced. Compared with the LPS-challenged group, after CU06-1004 treatment, the infiltration of immune cells was decreased in the BALF, and MPO activity in lung tissue was reduced. Similarly, in the CU06-1004 treatment group, pro-inflammatory cytokines were significantly inhibited in both BALF and serum. Evans-blue leakage was reduced, and the expression of junctional proteins was recovered in the CU06-1004 group. Adhesion molecules were downregulated and NF-κB activation was inhibited after CU06-1004 treatment. CONCLUSIONS: These results suggested that CU06-1004 had a therapeutic effect against LPS-induced ALI via alleviation of the inflammatory response and protection of vascular integrity.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Long-term administration of CU06-1004 ameliorates cerebrovascular aging and BBB injury in aging mouse model.

BACKGROUND: Age-related changes in the cerebrovasculature, including blood-brain barrier (BBB) disruption and vascular dementia, are emerging as potential risks for many neurodegenerative diseases. Therefore, the endothelial cells that constitute the cerebrovasculature may play key roles in preventing brain injury. Our previous study showed that CU06-1004, an endothelial cell dysfunction blocker, prevented vascular leakage, enhanced vascular integrity in ischemic reperfusion injury, and promoted the normalization of tumor vasculature. Here, we evaluated the effects of CU06-1004 on age-related cerebrovascular functional decline in the aged mouse brain. RESULTS: In this study, we investigated the protective effects of CU06-1004 against oxidative stress-induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with hydrogen peroxide (H2O2) to establish an oxidative stress-induced model of cellular injury. Compared with H2O2 treatment alone, pretreatment of HBMECs with CU06-1004 considerably reduced oxidative stress-induced cytotoxicity, reactive oxygen species generation, senescence-associated ß-galactosidase activity, senescence marker expression, and the expression levels of inflammatory proteins. Based on the observed cytoprotective effects of CU06-1004 in HBMECs, we examined whether CU06-1004 displayed protective effects against cerebrovascular aging in mice. Long-term administration of CU06-1004 alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged mouse brain. CU06-1004 supplementation also reduced the extravasation of plasma IgG by improving BBB integrity in the aged mouse brain, associated with reductions in neuronal injury. A series of behavioral tests also revealed improved motor and cognitive functions in aged mice that received long-term CU06-1004 administration. CONCLUSIONS: These findings suggest that CU06-1004 may represent a promising therapeutic approach for delaying age-related cerebrovascular impairment and improving cognitive function in old age.

The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Oral administration of CU06-1004 attenuates vascular permeability and stabilizes neovascularization in retinal vascular diseases.

Retinal vascular diseases are the leading cause of blindness worldwide. These diseases have common disease mechanisms including vascular endothelial growth factor (VEGF) signaling, hypoxia, and inflammation. Treatment of these diseases with laser therapy, anti-VEGF injections and/or steroids has significantly improved clinical outcomes. However, these strategies do not address the underlying cause of the pathology and may have harmful side effects. Pathological processes that damage retinal vessels result in vascular occlusion and impairment of the barrier properties of retinal endothelial cells, leading to excessive vascular leakage. Therefore, a new therapeutic approach is needed for the treatment of retinal vascular disease. We were able to confirm that oral administration of CU06-1004, an endothelial dysfunction blocker, inhibited retinal vascular leakage induced by vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2). Interestingly, oral administration of CU06-1004 prevented excessive vascular leakage in the diabetic retinopathy model. In addition, CU06-1004 inhibited angiogenesis and confirmed vascular stabilization in the oxygen-induced retinopathy model and laser-induced CNV model. Taken together, CU06-1004 could be a potential therapeutic agent for the treatment of retinal vascular diseases.

ALD-R491 regulates vimentin filament stability and solubility, cell contractile force, cell migration speed and directionality.

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis.

Enhancing Mixing Performance in a Rotating Disk Mixing Chamber: A Quantitative Investigation of the Effect of Euler and Coriolis Forces.

Lab-on-a-CD (LOCD) is gaining importance as a diagnostic platform due to being low-cost, easy-to-use, and portable. During LOCD usage, mixing and reaction are two processes that play an essential role in biochemical applications such as point-of-care diagnosis. In this paper, we numerically and experimentally investigate the effects of the Coriolis and Euler forces in the mixing chamber during the acceleration and deceleration of a rotating disk. The mixing performance is investigated under various conditions that have not been reported, such as rotational condition, chamber aspect ratio at a constant volume, and obstacle arrangement in the chamber. During disk acceleration and deceleration, the Euler force difference in the radial direction causes rotating flows, while the Coriolis force induces perpendicular vortices. Increasing the maximum rotational velocity improves the maximum rotational displacement, resulting in better mixing performance. A longer rotational period increases the interfacial area between solutions and enhances mixing. Mixing performance also improves when there is a substantial difference between Euler forces at the inner and outer radii. Furthermore, adding obstacles in the angular direction also passively promotes or inhibits mixing by configuration. This quantitative investigation provides valuable information for designing and developing high throughput and multiplexed point-of-care LOCDs.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

A rare case of the IgE prozone phenomenon in IgE multiple myeloma.

IgE multiple myeloma is a rare subtype of myeloma, accounting for <0.1% of all myeloma cases. Difficulties in diagnosing and monitoring this rare myeloma type are recognized, including the need of heightened awareness for initial diagnosis, performing a reflex immunofixation test using an anti-IgE antisera, and recognizing the possibility of the prozone phenomenon. Here, we report a rare case of IgE multiple myeloma with the prozone phenomenon. This case was characterized by a paradoxical increase in IgE levels with a progressive increase in the dilution factor. Moreover, serial monitoring of IgE levels correlated with the trend in the serum free light chain ratio, especially when the monoclonal protein was no longer detectable. This case highlights the need for laboratory professionals to be vigilant about the occurrence of the prozone phenomenon in IgE multiple myeloma.

Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome.

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI ß, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.

Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean.

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.