Dot Blot Research Articles

NAT10 promotes the tumorigenesis and progression of laryngeal squamous cell carcinoma through ac4C modification of FOXM1 mRNA

ABSTRACT Laryngeal squamous cell carcinoma (LSCC), is a prevalent malignant tumor, belongs to the category of head and neck tumors. N-acetyltransferase 10 (NAT10) can alter mRNA stability through N4- acetylcytidine (ac4C) modification. This study aimed to make an investigation into the role of NAT10-mediated ac4C modification in the malignant processes of LSCC cells. The NAT10 expression in LSCC tissues and cells was detected RT-qPCR and western blot. The ac4C dot blot was performed to detect ac4C level. Besides, the cell viability, migration, and invasion abilities were detected by CCK-8 and transwell assays. AcRIP-qPCR was performed to measure the abundance of ac4C on FOXM1 mRNA. RIP and Luciferase reporter assays were performed to demonstrate the interaction between NAT10 and FOXM1. Finally, the xenograft model was established to explore the role of NAT10 in vivo. NAT1 levels were significantly increased in the LSCC tissues and cells. Knockdown of NAT10 could significantly suppress the proliferation, migration, and invasion of LSCC cells. Additionally, NAT10 recognized the ac4C-modified sites in the 3’-untranslated regions (3’ UTR) of forkhead box M1 (FOXM1) to enhance the ability of FOXM1 mRNA. Furthermore, FOXM1 overexpression reversed the suppressing effects of NAT10 knockdown on the proliferation, migration, and invasion of LSCC cells, according to the results of rescue assays. Finally, results of animal experiments showed that NAT10 promoted in vivo tumorigenesis of LSCC cells through upregulating FOXM1. Our current study demonstrated that NAT10-mediated ac4C modification of FOXM1 mRNA promoted the malignant processes of LSCC cells.

Molecular patterns of alpha-thalassemia in the kingdom of Saudi Arabia: identification of prevalent genotypes and regions with high incidence

BackgroundAlpha-thalassemia (α-thalassemia) is one of the most common monogenic diseases in Saudi Arabia and is associated with significant morbidity. Premarital testing programs in Saudi Arabia reduce the burden of hemoglobinopathy disorders, and ongoing monitoring is required. We aimed to explore the molecular nature of α-globin genes and identify the most common genotypes and regions with a high risk of α-thalassemia in Saudi Arabia.MethodsThis retrospective study was conducted between January 2021 and December 2022. Six hundred twenty-five samples from patients with microcytic hypochromic anemia in Saudi Arabia were analyzed using reverse dot blot hybridization (RDBH)-based multiplex-PCR, which screens for the known 21 mutations of α-globin genes.ResultsSeven mutations in the α-globin gene were identified in 88.96% (556) patients. The most frequent abnormality of a-globin genes was −α3.7 (62.3%), followed by α2IVS1(−5nt) (20.7%) and α2 polyA-1 (α2T.Saudi) (14.1%). Interestingly, α2 polyA-2 (α2T.Turkish) was identified in Saudi and presented with −MED, causing Haemoglobin H disease. The incidence of α-thalassemia in Saudi Arabia’s cities showed significant differences (P = 0.004). Jeddah City had the highest percentage of cases (25%), followed by Makkah (23%), Taif (13.3%), and Al-Ahassa (12.4%).ConclusionThe study provides current knowledge about the molecular nature of α- thalassemia, highlights the common genotypes that could contribute to disease occurrence in the Saudi population, and sheds light on Saudi regions with a high incidence. It also recommends further studies in a larger population and with differently composed molecular assays to verify these findings.

In vitro screening of ninety-five plant extracts for inhibition of Clostridium histolyticum collagenase and Bacillus polymyxa metalloprotease- analogous human MMPs

Introduction and Aim: Matrix metalloproteinases (MMPs) are involved in many human diseases. Medicinal plants and their different constituents are utilized as therapeutic agents. In the present work we report the total phenolics content and screening of ninety five plant extracts for inhibitory activities against Clostridium histolyticum collagenase (ChC) and Bacillus polymyxa metalloprotease (BpM). Materials and Methods: Total phenolics content from plant methanolic extracts were assessed using Folin-Ciocalteau assay. Screening of plant extracts for ChC and BpM inhibitory activities was performed using dot blot assay on X-ray film. Results: G. superba fruit was observed to have the highest phenolics (257.58 ± 0.75 mg GAE /g tissue) content while C. nurvala stem was observed to have the lowest phenolics (0.468 ± 0.003 mg GAE /g tissue) content. Thirty two plants showed ChC inhibitory activities while twenty seven plants showed BpM inhibitory activities. From those plants twenty five plants have common ChC and BpM inhibitory activities. Most of plant barks were found to have a high amount of total phenolics as compared to other plant tissues while the most of plant leaves were found to have low amounts of total phenolics. Most of barks were found to exhibit inhibitory activities as compared to other tissue. Only 6 different leaves were found to exhibit inhibitory activity out of 42 different leaves. None of the root extracts exhibited inhibitory activities. Conclusion: The inhibitory activity exhibiting plant extracts could be used as a significant source for the determination of novel drug candidates to treat MMPs related diseases.

Production of Anti-Recombinant Human Insulin Antibody and Validation by Indirect ELISA

Human insulin potential has become an interest and is important in maintaining the success of therapyin patients with the availability of chemical-based analytical methods, however, only a few have beenusing immunoassays. This study aimed to produce IgG polyclonal antibodies from rabbits immunizedwith 1 mg/mL rhINS subcutaneously and validated by indirect ELISA. Antibody was precipitated andfractioned on a HiTrap® Protein A HP column before being quantified with a UV spectrophotometerat λ 280 nm. The characterization was conducted by Dot Blot test on a BCIP-NBT substrate, as well asSDS-PAGE and Western Blot with polyacrylamide gel concentrations of 7.5% and 17.5%. Validationwas performed using solutions containing glycerol and m-cresol as matrices spiked with rhINS. Thelinearity test in the rhINS concentration range of 80.11-200.28 μg/mL (r = 0.99) showed the linearresult. The accuracy and precision obtained an average of 99.11%±5.01 and 3.91%, while the LODand LOQ were 22.05 μg/mL and 73.51 μg/mL, respectively. Human insulin was stable at 2-8oC for 24hours (α: 0.05, ANOVA). In conclusion, in-house produced IgG polyclonal antibodies and goat anti-IgG peroxidase conjugate can be used for routine testing of human insulin.

Production and characterization of egg yolk antibodies against the ZIKV NS2B expressed in Nicotiana benthamiana

The emergence of Zika virus (ZIKV) and its associated neonatal and congenital complications pose a threat to global health, particularly in tropical and subtropical regions with co-circulation of related flaviviruses and intense vector proliferation. Diagnosis of ZIKV by RT-PCR is limited to the viraemic phase and is not always accessible in low-income tropical settings, while serological tests often show cross-reactivity with other flaviviruses. Given the similarity of ZIKV symptoms to those of other arboviruses, but the different prognosis and risks, it is important to develop specific and accessible diagnostic tools. Egg yolk antibodies (IgY) were obtained from Leghorn laying hens immunized with recombinant ZIKV NS2B protein produced in agroinfiltrated Nicotiana benthamiana. After three immunizations, total IgY was recovered from the eggs by the 20% ammonium sulfate precipitation method. After characterisation by SDS-PAGE, dot blotting and ELISA, the IgY was adsorbed to dengue virus (DENV) from cell culture supernatants and tested for its ability to specifically detect ZIKV-positive sera samples. High yield and purity were observed on SDS-PAGE for polyclonal IgY, which reacted with NS2B at high titres in ELISA and detected both NS2B and ZIKV in dot blotting. However, a cross-reaction with DENV was observed and the anti-NS2B IgY was unable to discriminate ZIKV from DENV positive sera samples, even after adsorption with DENV. This is probably due to the phylogenetic relationship of the viruses and the shared identity of their proteins.

Scleromyxedema associated with macular edema and retinal hemorrhage.

We discuss a case of macular edema and retinal hemorrhage associated with scleromyxedema. A case report is presented. A 64-year-old male with history of deep vein thrombosis and pulmonary embolism presented with new onset rash in the setting of switching anticoagulation treatments. He developed blurred vision was found to have macular edema and dot blot retinal hemorrhages which improved with systemic and topical corticosteroids. Systemic autoimmune conditions including scleromyxedema should be considered in the workup of occult cystoid macular edema.

The bacterial tubulin homolog FtsZ generates electrical oscillations

FtsZ, a major cytoskeletal protein in all bacteria and archaea, forms a ring that directs cytokinesis. Bacterial FtsZ is considered the ancestral homolog of the eukaryotic microtubule (MT)-forming tubulins, sharing GTPase activity and the ability to assemble into protofilaments, rings, and sheets, but not MTs. Previous studies from our laboratory demonstrated that structures of isolated brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. No information about whether the prokaryotic tubulins may share similar properties is available. Here, we obtained by ammonium sulfate precipitation an enriched protein fraction of the endogenous FtsZ from wild-type Escherichia coli ATCC 25922 without any transfection or overexpression of the protein. As revealed by electron microscopy, FtsZ was detected by dot blot analysis and immunofluorescence that assembled into filaments and sheets in a polymerization buffer. We used the patch-clamp technique to explore the electrical properties of sheets of FtsZ and bacterial cells. Electrical recordings at various holding potentials ranging from ±200 mV showed a complex oscillatory behavior, with several peak frequencies between 12 and 110 Hz in the power spectra and a linear mean current response. To confirm the oscillatory electrical behavior of FtsZ we also conducted experiments with commercial recombinant FtsZ, with similar results. We also detected, by local field potentials, similar electrical oscillations in K+-depolarized pellets of E. coli cultures. FtsZ oscillations had a wider range of frequency peaks than MT sheets from eukaryotic origin. The findings indicate that the bacterial cytoskeleton generates electrical oscillators that may play a relevant role in cell division and unknown signaling mechanisms in bacterial populations.

Acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone targeting cellular JUN proto-oncogene, AP-1 transcription factor subunit inhibits head and neck squamous cell carcinoma progression.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide with a survival rate below fifty percent. Addressing meager therapeutic options, a series of small molecule inhibitors were screened for antitumor efficacy. The most potent analog, acryl-3,5-bis(2,4-difluorobenzylidene)-4-piperidone (DiFiD; A-DiFiD), demonstrated strong cellular JUN proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (JUN, c-Jun) antagonism. c-Jun, an oncogenic transcription factor, promotes cancer progression, invasion, and adhesion; high (JUN) mRNA expression correlates with poorer HNSCC survival. Four new small molecules were generated for cytotoxicity screening in HNSCC cell lines. A-DiFiD-treated HNSCC cells were assessed for cytotoxicity, colony formation, invasion, migration, and adhesion. Dot blot array was used to identify targets. Phospho-c-Jun (p-c-Jun) expression was analyzed using immunoblotting. The Cancer Genome Atlas (TCGA) head and neck cancer datasets were utilized to determine overall patient survival. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) datasets interfaced with University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) were analyzed to determine protein levels of c-Jun in HNSCC patients and correlate levels with patient. Of the small molecules tested, A-DiFiD was the most potent in HNSCC lines, while demonstrating low half-maximal drug inhibitory concentration (IC50) in non-malignant Het-1A cells. Additionally, A-DiFiD abrogated cell invasion, migration, and colony formation. Phospho-kinase in vitro array demonstrated A-DiFiD reduced p-c-Jun. Likewise, a time dependent reduction in p-c-Jun was observed starting at 3 min post A-DiFiD treatment. TCGA Firehose Legacy vs. recurrent and metastatic head and neck cancer reveal a nearly 3% DNA amplification in recurrent/metastatic tumor compared to below 1% in primary tumors that had no lymph node metastasis. CPTAC analysis show higher tumor c-Jun levels compared to normal. Patients with high JUN expression had significantly reduced 3-year survival. A-DiFiD targets c-Jun, a clinical HNSCC driver, with potent anti-tumor effects.

Prediction, Synthesis and Evaluation of a Synthetic Peptide as an Enzyme-Linked Immunosorbent Assay (ELISA) Candidate for Screening of Bovine Antibodies against Theileria annulata.

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen's kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata.

Immunized mice naturally process in silico-derived peptides from the nucleocapsid of SARS-CoV-2

BackgroundThe nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an excellent immunogen that promotes the production of high-titer antibodies. N protein-derived peptides identified using a bioinformatics approach can potentially be used to develop a new generation of vaccines or diagnostic methods for detecting SARS-CoV-2 and its variants. However, further studies must demonstrate their capacity to be naturally processed by the immune system.ObjectiveWe aimed to examine the in vivo processing and recognition of in silico-identified peptides using the serum of immunized animals with the complete protein.MethodsRecombinant N (Nrec) protein was subcutaneously administered to six Balb/c mice. Enzyme-linked immunosorbent assay (ELISA), western blotting, dot blotting, and immunoprecipitation were performed to evaluate the recognition of the complete protein and in silico-derived peptides.ResultsThe serum of immunized mice recognized ~ 62.5 ng/µL of Nrec with high specificity to linear and conformational epitopes. Dot blot analysis showed that peptides Npep2 and Npep3 were the most reactive.ConclusionOur data confirm the high immunogenicity of the SARS-CoV-2 N protein and provide evidence on the antigenicity of two peptides located in the N-arm/RNA-binding domain (Npep2) and oligomerization domain/C-tail (Npep3), considered the biologically active site of the N protein.

Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin–phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

Construction of L-type lectin displaying Saccharomyces cerevisiae for Vibrio parahaemolyticus agglutination.

The utilization of Aga1P anchor protein in the display system for expressing heterologous proteins on the surface of Saccharomyces cerevisiae has been shown to be an ideal approach. This system has the ability to improve the expression of target proteins beyond the cell surface, resulting in increased activity and stability of the expression system. Recent studies have demonstrated that a new L-type lectin from Litopenaeus vannamei (LvLTLC1) has been found to possess the capability of agglutinating Vibrio parahaemolyticus, a pathogen responsible for causing acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, LvLTLC1 protein was designed to be expressed on the surface of S. cerevisiae via Aga1P anchor. The expression of LvLTLC1 protein on the surface of S. cerevisiae::pYIP-LvLTLC1-Aga1P was confirmed through the use of analytical techniques including SDS-PAGE, dot blot, and fluorescent immunoassay with LvLTC1-specific antibody. Subsequently, the newly generated yeast strain was evaluated for its ability to agglutinate V. parahaemolyticus and A. hydrophila. The obtained results indicated that S. cerevisiae expressing LvLTLC1 protein on its surface had the ability to agglutinate both AHPND-causing V. parahaemolyticus and A. hydrophila. This newly generated yeast strain could be served as a feed supplement for controlling bacteria in general and AHPND in particular.

Dot blot analysis indicates synapsin proteins I, II, and III are present in mouse sperm

Dot blot analysis indicates synapsin proteins I, II, and III are present in mouse sperm

IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway

ABSTRACT Background: N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA. However, its role in non-small cell lung cancer (NSCLC) has not been completely elucidated. Objective: To explore whether methyltransferase like 3 (METTL3) in cancer associated fibroblasts (CAFs) affects the secretion of IL-18, which drives NSCLC cells to regulate PD-L1-mediated immunosuppression via the nuclear factor kappa B (NF-κB) pathway. Methods: Histopathological features of NSCLC tissues were identified by H&E and IHC staining. The levels of m6A writers (METTL3), IL-18 and NF-κB pathway related genes were assessed. The quantity of CD8+ T cells was evaluated by flow cytometry (FCM). The direct binding relationship between METTL3 and IL-18 mRNA was detected by RIP assay and RNA pulldown and confirmed by dual – luciferase reporter assay. The level of RNA m6A was detected by RNA m6A dot blot and meRIP assays. A heterotopic implantation model of NSCLC was established in NOD-SCID mice for further explore the effect of CAF derived METTL3 on immunosuppression of NSCLC in vivo. Results: Our results illustrated that METTL3 was down-regulated in CAFs, and CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. Subsequently, we found that IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC, and IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. In vivo, METTL3 knockdown-derived CAFs accelerated immunosuppression of NSCLC. Conclusion: IL-18 served as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

Cytokine expression profile of native heart valves obtained from patients with infective endocarditis

Infectious endocarditis is characterized by dysfunction of heart valves and contribute significantly to the cardiovascular morbidity and mortality worldwide, especially in low- and middle-income countries. Immune response is playing the important role in the pathophysiology of this disease. This work was aimed to study the local cytokine profile in native heart valves obtained from the patients with infectious endocarditis. Cytokine profiling were performed in biopsies of native heart valves explanted from 4 patients with infective endocarditis (experimental group) and 10 patients with rheumatic heart disease (comparison group) by dot blotting using the Proteome Profiler™ Human Cytokine Array Kit (ARY005B). The results of dot blotting were validated by the gene expression profiling using quantitative polymerase chain reaction. MIF, PAI-1, ICAM-1 and CXCL12 were found in the native heart valves explanted from both infective endocarditis and rheumatic heart disease patients. Upon a semi-quantitative assessment, the heart valves explanted from the infective endocarditis patients were characterized by 4-fold increased secretion of PAI-1 and twofold decreased secretion of ICAM-1 and CXCL12 compared to the heart valves ffected by rheumatic heart disease. MIF was expressed on similar levels in the both studied groups. IL-1ra, IL-6, IL-8, IL-16, CCL4, CCL5 and CXCL1 were detected only in heart valves affected by infectious endocarditis. At the gene expression level, MIF, IL6, IL8 genes were upregulated and PAI1, IL1RA, CXCL1 genes were downregulated in heart valves explanted from infectious endocarditis patients compared to the subjects affected by rheumatic heart disease. Native heart valves in cases of infectious endocarditis are characterized by nonspecific local inflammatory response associated with pathogenic bacteremia, along with active neovascularization. The data obtained can help to better understand fundamental pathogenetic mechanisms of infectious endocarditis.

METTL3-Mediated RNA m6A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2.

N6-methyladenosine (m6A) is a commonly occurring modification of mRNAs, catalyzed by a complex containing methyltransferase like 3 (METTL3). Our research aims to explore how METTL3-dependent m6A modification affects the functions of retinal endothelial cells (RECs). An oxygen-induced retinopathy (OIR) mouse model was established, and RECs were isolated using magnetic beads method. Human retinal microvascular endothelial cells (HRMECs) were treated with normoxia (21% O2) or hypoxia (1% O2). Dot blot assay determined m6A modification levels. Quantitative RT-PCR and Western blot detected the mRNA and protein expression levels of the target candidates, respectively. Genes were knocked down by small interfering RNA transfection. Matrigel-based angiogenesis and transwell assays evaluated the abilities of endothelial tube formation and migration, respectively. Methylated RNA immunoprecipitation-qPCR determined the levels of m6A modification in the target genes. The m6A modification levels were significantly upregulated in the retinas and RECs of OIR mice. Exposure to hypoxia significantly elevated both METTL3 expression and m6A modification levels in HRMECs. METTL3 knockdown curtailed endothelial tube formation and migration in vitro under both normoxic and hypoxic conditions. Concurrently, this knockdown in HRMECs resulted in reduced m6A modification levels of MMP2 and TIE2 transcripts, subsequently leading to a decrease in their respective protein expressions. Notably, knockdown of MMP2 and TIE2 also markedly inhibited the angiogenic activities of HRMECs. METTL3-mediated m6A modification promotes the angiogenic behaviors of RECs by targeting MMP2 and TIE2, suggesting its significance in retinal angiogenesis and METTL3 as a potential therapeutic target.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Inhibition of METTL3 alleviated LPS-induced alveolar epithelial cell apoptosis and acute lung injury via restoring neprilysin expression

AimsTo investigate the role and mechanisms of methyltransferase-like 3 (METTL3) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Main methodsLPS intratracheally instillation was applied in alveolar epithelial cell METTL3 conditional knockout (METTL3-CKO) mice and their wild-type littermates. In addition, METTL3 inhibitor STM2457 was used. LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP. Key findingsMETTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. Knockdown of METTL3 alleviated, while overexpression of METTL3 exacerbated LPS-induced apoptosis. LPS treatment reduced neprilysin expression, the intervention of neprilysin expression negatively regulated apoptosis without affecting METTL3 expression, and mitigated the promoting effect of METTL3 on LPS-induced apoptosis. Additionally, METTL3 could bind to the mRNA of neprilysin, and reduce its expression. SignificanceOur findings revealed that inhibition of METTL3 could exert anti-apoptosis and ALI-protective effects via restoring neprilysin expression.

Long-Read Sequencing Identified a Large Novel δ/β-Globin Gene Deletion in a Chinese Family

Objective. Increasingly rare thalassemia has been identified with the advanced use of long-read sequencing based on long-read technology. Here, we aim to present a novel δ/β-globin gene deletion identified by long-read sequencing technology. Methods. Enrolled in this study was a family from the Quanzhou region of Southeast China. Routine blood analysis and hemoglobin (Hb) capillary electrophoresis were used for hematological screening. Genetic testing for common α- and β-thalassemia was carried out using the reverse dot blot hybridization technique. Long-read sequencing was performed to detect rare globin gene variants. Specific gap-polymerase chain reaction (gap-PCR) and/or Sanger sequencing were further used to verify the detected variants. Results. None of the common α- and β-thalassemia mutations or deletions were observed in the family. However, decreased levels of MCV, MCH, and abnormal Hb bands were observed in the family members, who were suspected as rare thalassemia carriers. Further, long-read sequencing demonstrated a large novel 7.414 kb deletion NG_000007.3:g.63511_70924del partially cover HBB and HBD globin genes causing delta-beta fusion gene in the proband. Parental verification indicated that the deletion was inherited from the proband’s father, while none of the globin gene variants were observed in the proband’s mother. In addition, the novel δ/β-globin gene deletion was further verified by gap-PCR and Sanger sequencing. Conclusion. In this study, we first present a large novel δ/β-globin gene deletion in a Chinese family using long-read sequencing, which may cause δβ-thalassemia. This study further enhances that long-read sequencing would be applied as a sharp tool for detecting rare and novel globin gene variants.

Light-mediated protein functionalization of photoclickable hydrogel interface for selective cell capture and dot blotting assay

The construction of versatile functional hydrogel interfaces holds promising prospects in biosensing and bioengineering. Herein, we introduced a light-induced protein conjugation strategy for on-demand surface modification of hydrogel interface based on the photoclick cyclization between primary amine and o-nitrobenzyl alcohol. We achieved the on-demand protein conjugation by grafting the molecular plugin, 4-(hydroxymethyl)-3-nitrobenzoic acid (HNBA), onto the hydrogel surface, followed by the mask-aided photoclick reaction with the protein of interest. This method enables the creation of protein patterns on hydrogel interface with a lower limit of pattern width at ∼70 μm. With this method, we demonstrated the surface engineering of epidermal growth factor (EGF) on hydrogel interface for selective capture of EGF receptor-positive cancer cells with an efficiency over 80%. Moreover, we applied the mask-aided photoclick conjugation method for antigen capture and developed a photoclickable hydrogel interface-based dot blotting assay. Due to the high-efficient antigen capture of photoclick conjugation, the photoclickable hydrogel interface-based dot blotting assay shows improved sensitivity for antigen detection with a limit of detection as 0.065 ng. We believed that this light-induced protein conjugation method holds the potential as a robust strategy for the construction of bioactive hydrogel interfaces for various bio-related applications.