Polymerase Chain Reaction Assay Research Articles

Alternative splicing-related long noncoding RNA ANRIL facilitates hepatocellular carcinoma by targeting the miR-199a-5p/SRSF1 axis and impacting Anillin.

Hepatocellular carcinoma (HCC) is characterized by aberrant alternative splicing (AS), which plays an important part in the pathological process of this disease. However, available reports about genes and mechanisms involved in AS process are limited. Our previous research has identified ANRIL as a long noncoding RNA related to the AS process of HCC. Here, we investigated the exact effect and the mechanism of ANRIL on HCC progress.The ANRIL expression profile was validated using the real-time quantitative polymerase chain reactionassay. The western blot analysis and IHC assay were conducted on candidate targets, including SRSF1 and Anillin. The clinicopathological features of 97 patients were collected and analyzed. Loss-of and gain-of-function experiments were conducted. The dual-luciferase reporter assay was applied to verify the interaction between ANRIL, miR-199a-5p, and SRSF1.Anomalous upregulation of ANRIL in HCC was observed, correlating with worse clinicopathological features of HCC. HCC cell proliferation, mobility, tumorigenesis, and metastasis were impaired by depleting ANRIL. We found that ANRIL acts as a sponger of miRNA-199a-5p, resulting in an elevated level of its target protein SRSF1. The phenotypes induced by ANRIL/miR-199a-5p/SRSF1 alteration are associated with Anillin, a validated HCC promoter.ANRIL is an AS-related lncRNA promoting HCC progress by modulating the miR-199a-5p/SRSF1 axis. The downstream effector of this axis in the development of HCC is Anillin.

Hematological and plasma profiles and ticks and tick-borne pathogens in wild Formosan black bears (Ursus thibetanus formosanus)

BackgroundThe endangered Formosan black bear (Ursus thibetanus formosanus) is the largest native carnivorous mammal in Taiwan. Diseases, poor management, illegal hunting, and habitat destruction are serious threats to the survival of bear populations. However, studies on the impact of diseases on bear populations are limited. Therefore, this study aimed to establish a database of the hematological and plasma profiles of free-ranging Formosan black bears and investigate the occurrence of ectoparasites, blood parasites, and vector-borne pathogens.MethodsFormosan black bears were captured in Yushan National Park (YNP) and Daxueshan Forest Recreation Area (DSY) in Taiwan. Blood samples were collected from each bear for hematological analysis and plasma biochemistry using a hematology analyzer. Parasites and pathogens were detected using a thin blood smear with Wright–Giemsa staining and polymerase chain reaction (PCR) assay. Additionally, macroscopic ectoparasites were collected from bears to detect blood parasites and other pathogens. Moreover, the relationships between the bear variables (sex, age, and occurrence of parasites or pathogens), ectoparasites, and infectious agents were also analyzed.ResultsIn all, 21 wild bears (14 in YNP and 7 in DSY) were captured and released during the satellite tracking studies. Hematological analysis and plasma biochemistry indicated significant differences in white blood cells (WBC), segments, creatine kinase (CK), and lactate dehydrogenase (LDH) levels between foot snare and culvert-captured bears. Additionally, there were significant differences in total plasma protein (TPP), creatinine, Ca2+, Mg2+, and K+ levels between male and female bears. Moreover, pathogen-infected bears had significantly higher erythrocyte sedimentation rate (ESR; 30 min and 1 h) and globulin levels than uninfected bears. In total, 240 ticks were collected from 13 bears, among which eight adult tick species were identified, including Haemaphysalis flava, Haemaphysalis hystricis, Amblyomma testudinarium, Ixodes ovatus, Dermacentor taiwanensis, Haemaphysalis longicornis, Ixodes acutitarsus, Amblyomma javanense, and nymphs belonging to Haemaphysalis spp. PCR revealed that 13 (61.90%) and 8 (38.10%) bears harbored Hepatozoon ursi and Babesia DNA, respectively. Among the ticks examined, 157 (65.41%) and 128 (53.33%) samples were positive for H. ursi and Babesia, respectively.ConclusionsTo the best of our knowledge, this is the first study to establish a database of the hematological and plasma profiles of wild Formosan black bears and investigate ectoparasite infestation and Hepatozoon and Babesia spp. infection. In conclusion, these findings may serve as a reference for monitoring the health and population of locally endangered bears.Graphical

Molecular mechanisms of poor osseointegration in irradiated bone: In vivo study in a rat tibia model.

Radiotherapy is associated with cell depletion and loss of blood supply, which are linked to compromised bone healing. However, the molecular events underlying these effects at the tissue-implant interface have not been fully elucidated. This study aimed to determine the major molecular mediators associated with compromised osseointegration due to previous exposure to radiation. Titanium implants were placed in rat tibiae with or without pre-exposure to 20 Gy irradiation. Histomorphometric, biomechanical, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay analyses were performed at 1 and 4 weeks after implantation. The detrimental effects of irradiation were characterized by reduced bone-implant contact and removal torque. Furthermore, pre-exposure to radiation induced different molecular dysfunctions such as (i) increased expression of pro-inflammatory (Tnf) and osteoclastic (Ctsk) genes and decreased expression of the bone formation (Alpl) gene in implant-adherent cells; (ii) increased expression of bone formation (Alpl and Bglap) genes in peri-implant bone; and (iii) increased expression of pro-inflammatory (Tnf) and pro-fibrotic (Tgfb1) genes in peri-implant soft tissue. The serum levels of pro-inflammatory, bone formation and bone resorption proteins were greater in the irradiated rats. Irradiation causes the dysregulation of multiple biological activities, among which perturbed inflammation seems to play a common role in hindering osseointegration.

Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy

The nuclear receptor Nur77 plays paradoxical roles in numerous cancers. However, whether Nur77 inhibits esophageal squamous cell carcinoma (ESCC) growth and affects immunological responses against ESCC has not been determined. The functional role of Nur77 in ESCC was investigated in this study using human ESCC cell lines, quantitative real-time polymerase chain reaction (PCR), cell proliferation and colony formation assays, flow cytometry analysis, western blotting and animal models. The target gene controlled by Nur77 was verified using dual-luciferase reporter assays, chromatin immunoprecipitation analysis and functional rescue experiments. To examine the clinical importance of Nur77, 72 human primary ESCC tissues were subjected to immunohistochemistry. Taken together, these findings showed that, both in vitro and in vivo, Nur77 dramatically reduced ESCC cell growth and triggered apoptosis. Nur77 directly interacts with the interferon regulatory factor 1 (IRF1) promoter to inhibit its activity in ESCC. Pharmacological induction of Nur77 using cytosporone B (CsnB) inhibited ESCC cell proliferation and promoted apoptosis both in vitro and in vivo. Furthermore, CsnB increased CD8+ T-cell infiltration and cytotoxicity to inhibit the formation of ESCC tumors in an immunocompetent mouse model. In ESCC tissues, Nur77 expression was downregulated, and IRF1 expression was increased; moreover, their expression levels were negatively related. IRF1 and Nur77 were strongly correlated with overall survival. These findings suggested that Nur77 targets and regulates the IRF1/PD-L1 axis to serve as a tumor suppressor in ESCC.Graphical abstract of the regulatory mechanism of Nur77 overexpression downregulates IRF1 in the inhibition of ESCC progression and enhance anti-PD-1 therapy efficacy.

Association of the retinol to all-trans retinoic acid pathway with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex group of neurodevelopmental disorders. Research has highlighted a close association between the retinoic acid (RA) signaling pathway and ASD. This study investigates alterations in the vitamin A (VA, retinol) to RA metabolic pathway in children with ASD and speculates on the underlying reasons for these changes. We propose a subtype characterized by downregulated RA signaling in ASD, laying the groundwork for precise diagnosis and treatment research. We included 489 children with ASD and 280 typically developing (TD) children. Those with ASD underwent evaluations of core symptoms and neuro-developmental levels, which were conducted by professional developmental behavior physicians using assessment scales. Serum VA and all-trans RA (atRA) levels were determined by high-performance liquid chromatography and ultra-high-performance liquid chromatography-tandem mass spectrometry. The expression levels and concentrations of enzyme molecules such as retinol dehydrogenase 10 were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Children with ASD exhibited reduced serum atRA, accompanied by a downregulation of atRA synthesis enzymes. The reduction in serum atRA levels was linked not only to VA levels but also to the aberrant expression of metabolic enzymes responsible for atRA. Furthermore, the serum atRA levels in children with ASD were more strongly correlated with core symptoms and neurodevelopmental levels than VA levels. Children with ASD exhibited a dual regulation of reduced serum atRA levels, influenced by both VA levels and abnormal expression of atRA metabolic enzymes.

Application of Novel Gill Cell Line from Lates calcarifer for Recognizing Metals Using Probes.

Lates calcarifer (Bloch) is a potential candidate fish species for culture in marine and brackishwater. A continuous gill cell line was derived from L. calcarifer by the explant culture method and has been passaged for 132 times, in Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) at 28°C. The cells showed a rate of recovery between 90 and 95% after being successfully cryopreserved at various passage levels and formed monolayer in 2-3days without any morphological changes. Immunophenotypic analysis of the SBG cell line revealed that they are of epithelial origin. Polymerase chain reaction assay using mitochondrial 12S rRNA primer specific to L. calcarifer was used to confirm the authenticity of the established gill cell line origin from seabass. The transfection efficiency was evaluated in Seabass Gill (SBG) cell line using pEGFP-N1 and Lipofectamine™ 3000. Transfection efficiency was found to be between 13 and 16%. The cytotoxicity of three different metal detecting probes was evaluated by MTT and Alamar blue assays to determine safe concentration. The result revealed that SBG cell line can be applied for recognition of metals using probes. The current study established, for the first time, a gill-derived cell line (SBG) from Lates calcarifer and its application for the detection of intracellular indium, mercury, and lutetium ions by specific fluorescent probes.

HLA-DRB5 promotes immune thrombocytopenia via activating CD8+ T cells.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet (PLT) count and a high risk of bleeding, the clinical treatment for which still needs to be upgraded. Based on the critical role of human leukocyte antigen class II heterodimer β5 (HLA-DRB5) in immune system, we herein investigated its effect on ITP. ITP murine models were established by the injection of guinea pig anti-mouse platelet serum (GP-APS), and the PLT of mouse peripheral blood was counted during the modeling. Quantitative real-time reverse transcription polymerase chain reaction, western blot and immunofluorescence assay was performed to quantify expressions of HLA-DRB5, major histocompatibility complex II (MHC-II) and co-stimulatory molecules (CD80, CD86). Flow cytometry was conducted to analyze the percentage of CD8+ T cells. As a result, the PLT count was decreased in mouse peripheral blood. Expressions of HLA-DRB5, MHC-II and co-stimulatory molecules, as well as the percentage of CD8+ T cells were elevated in peripheral blood of ITP mice. HLA-DRB5 knockdown mitigated ITP by increasing peripheral PLT level, downregulating expressions of MHC-II and co-stimulatory molecules and inactivating CD8+ T cells. Collectively, the downregulation of HLA-DRB5 restores the peripheral PLT count in ITP mice by reducing MHC-II-mediated antigen presentation of macrophages to inhibit the activation of CD8+ T cells.

HIV-1 RNA in extracellular vesicles is associated with neurocognitive outcomes

Human immunodeficiency virus type-1 (HIV-1) is responsible for significant mortality and morbidity worldwide. Despite complete control of viral replication with antiretrovirals, cells with integrated HIV-1 provirus can produce viral transcripts. In a cross-sectional study of 84 HIV+ individuals of whom 43 were followed longitudinally, we found that HIV-1 RNAs are present in extracellular vesicles (EVs) derived from cerebrospinal fluid and serum of all individuals. We used seven digital droplet polymerase chain reaction assays to evaluate the transcriptional status of the latent reservoir. EV-associated viral RNA was more abundant in the CSF and correlated with neurocognitive dysfunction in both, the cross-sectional and longitudinal studies. Sequencing studies suggested compartmentalization of defective viral transcripts in the serum and CSF. These findings suggest previous studies have underestimated the viral burden and there is a significant relationship between latent viral transcription and CNS complications of long-term disease despite the adequate use of antiretrovirals.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Prediction of severe preeclampsia and intrauterine growth restriction based on serum placental exosome miR-520a-5p levels during the first-trimester.

To assess the predictive capabilities of serum exosomal levels of micro-RNA-520a-5p (miR-520a-5p) concerning the occurrence of severe preeclampsia (sPE) and fetal growth restriction (FGR) during the first trimester of pregnancy. During the period spanning from October 2020 to October 2021, serum samples were procured from the first trimester and subsequently preserved by freezing at -80 ℃. These samples were obtained from 105 pregnant women in a nested case-control study. This cohort consisted of individuals who later developed sPE (sPE group, n = 35) and FGR (FGR group, n = 35) during the third trimester. Additionally, 35 women with normal blood pressure were denoted as normal pregnancy group. Serum samples from the first trimester were retrieved from all groups for further analysis after thawing. Exosomes were extracted from the serum samples collected during the first trimester and examined using transmission electron microscopy, western blot, and nanoparticle tracking analysis. Additionally, the determination of their placental origin was also established during the course of the study. Exosome miR-520a-5p levels were measured using real-time quantitative polymerase chain reaction assays, primarily involving quantitative reverse transcription polymerase chain reactions. Fetal placental tissues from the 3 groups were collected shortly after birth, and miR-520a-5p expression was measured using real-time quantitative polymerase chain reaction. Serum placental exosomes and fetal placental tissues were compared for miR-520a-5p levels. Placental trophoblasts were identified as the source of serum exosomes in all 3 groups. It was found that serum placental exosomes exhibited lower levels of miR-520a-5p in both the sPE and FGR groups when compared to the normal pregnancy group. This finding was consistent with observations made in postpartum placental tissues. The predictive accuracy for sPE using miR-520a-5p levels in serum placental exosomes during the first trimester was notably higher (area under the receiver operating characteristic curve = 0.806, P <.05) compared to the prediction of FGR (area under the receiver operating characteristic curve = 0.628, P <.05). Placenta-derived exosomes can be extracted from maternal serum during the first trimester of pregnancy and miR-520a-5p detected from the exosomes. The downregulation of miR-520a-5p serves as a more predictive indicator for the subsequent development of sPE compared to predicting FGR.

Fine mapping and candidate gene analysis of CRA8.1.6, which confers clubroot resistance in turnip (Brassica rapa ssp. rapa)

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255–12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain’s C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

Assessment of fungal species in some spices using molecular study with detection of their mycotoxins using chromatography analysis.

Spices are food flavouring agents that are highly used in Iraq. However, they may be contaminated by toxicogenic fungi and subsequent production of mycotoxins. The aim of this study was to investigate the contamination of commonly used spices with fungi using polymerase chain reaction (PCR) assay and to detect fungal mycotoxin using high-performance liquid chromatography. Thirty-five spices (seven samples from each black pepper, red pepper, turmeric, cumin and ginger) were cultured on an appropriate medium to identify various fungi species. Later on, the toxigenicity of Aspergillus flavus and Aspergillus niger was determined using a PCR assay. The fungal mycotoxins, including aflatoxins and ochratoxins, were then determined through a high-performance liquid chromatography using the validated Quick, Easy, Cheap, Effective, Rugged, and Safe (QuECHERS) method. Aspergillus species were the predominantly isolated fungi, followed by Penicillium and Fusarium. The PCR results indicate the high toxigenicity of A. flavus as 85.7% of the strains had aflQ/aflR genes and 79% had PKS15KS/PKS15C-MeT genes. Regarding mycotoxin contamination in spices, the highest rates of aflatoxins and ochratoxins were found in black pepper (5.913 μg/kg) and red chilli (6.9055 μg/kg), respectively. Spices are susceptible substrates for the growth of mycotoxigenic fungi. Thus, regular effective surveillance and quality control procedures are highly recommended.

Evaluation of Gram Stain-Guided Antibiotic Therapy for Methicillin-Resistant Staphylococcus aureus Pneumonia in Intensive Care Unit Patients.

Background: Despite high negative predictive values (NPVs) seen with methicillin-resistant Staphylococcus aureus (MRSA) nares polymerase chain reaction (PCR) assays, utilization of both respiratory sample Gram stain and MRSA nares PCR in patients with pneumonia may contribute to overuse of laboratory resources. The purpose of this study was to evaluate if a Gram stain demonstrating no Gram-positive organisms from a respiratory sample is sufficient to allow for de-escalation of vancomycin therapy. Methods: This single center study retrospectively identified intensive care unit (ICU) patients started on vancomycin for presumed pneumonia at University of Wisconsin (UW) Health in Madison, WI between August 2022 and March 2023. Patients with respiratory sample demonstrating no Gram-positives on Gram stain met inclusion criteria if the sample was ordered within 24 h of vancomycin initiation. The primary outcome was NPV of respiratory sample Gram stain demonstrating no Gram-positive organisms with respect to MRSA detection of the respiratory culture. Secondary outcomes included the NPV of combined MRSA nares PCR plus respiratory sample Gram stain, and difference in time to event in patients that had both a respiratory sample and MRSA nares PCR ordered. Results: A total of 370 patients were screened for study eligibility; of which 99 patients met inclusion criteria. NPV of respiratory sample Gram stain was 99% for MRSA culture. The combined NPV of respiratory sample Gram stain plus MRSA nares PCR was 98.9% for MRSA culture (n = 88). Respiratory sample was ordered 2.3 h faster compared to MRSA nares PCR (4.3 vs 6.6 h, P = .036). Respiratory sample Gram stain resulted 4.5 h faster compared to MRSA nares PCR (10.7 vs 15.2 h, P = .002). Conclusion: Respiratory sample Gram stains demonstrating no Gram-positive organisms may be used to de-escalate vancomycin and deprioritize the use of MRSA nares PCR.

Interrogating the estrogen-mediated regulation of adrenocortical Klotho expression using ovariectomized albino rat model exposed to repeated restraint stress.

Reproductive aging is associated with altered stress response and many other menopausal symptoms. Little is known about the adrenal expression of the anti-aging protein Klotho or how it is modulated by estrogen in ovariectomized stressed rats. Fifty-six Wistar female rats were assigned into seven equal groups. Sham-operated (Sham), sham stressed (Sham/STS), ovariectomized (OVR), ovariectomized stressed (OVR/STS), ovariectomized stressed rosiglitazone-treated (OVR/STS/R), ovariectomized stressed estrogen-treated (OVR/STS/E), and ovariectomized stressed estrogen/GW9662 co-treated (OVR/STS/E/GW) groups. All stressed rats were subjected daily to a one-hour restraint stress test for 19days.At the end of the experiment, blood was collected for serum corticosterone (CORT) analysis. Adrenal tissues were obtainedand prepared for polymerase chain reaction (PCR) assay, hematoxylin and eosin (H&E), immunohistochemistry-based identification of Klotho and PPAR-γ, and Oil Red O (ORO) staining. The rise in serum CORT was negligible in the OVR/STS group, in contrast to the Sham/STS group. The limited CORT response in the former group was restored by estrogen and rosiglitazone and blocked by estrogen/GW9226 co-administration. ORO-staining revealed a more evident reduction in the adrenal fat in the OVR/STS group, which was reversed by estrogen and counteracted by GW. Also, there was a comparable expression pattern of Klotho and PPAR-γ in the adrenals. The adrenal Klotho decreased in the OVR/STS group, but was reversed by estrogen treatment. GW9226/estrogen co-treatment interfered with the regulatory effect of estrogen on Klotho. The study suggested modulation of the adrenal Kotho expression by estrogen, in the ovariectomized rats subjected to a restraint stress test. This estrogen-provided adrenal protection might be mediated by PPAR-γ activation.

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress.

This study investigates the impact of fluctuating lipid levels on endothelial dysfunction. Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay. Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression. Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

AnoPrimer: Primer Design in malaria vectors informed by range-wide genomic variation

The major malaria mosquitoes, Anopheles gambiae s.l and Anopheles funestus, are some of the most studied organisms in medical research and also some of the most genetically diverse. When designing polymerase chain reaction (PCR) or hybridisation-based molecular assays, reliable primer and probe design is crucial. However, single nucleotide polymorphisms (SNPs) in primer binding sites can prevent primer binding, leading to null alleles, or bind suboptimally, leading to preferential amplification of specific alleles. Given the extreme genetic diversity of Anopheles mosquitoes, researchers need to consider this genetic variation when designing primers and probes to avoid amplification problems. In this note, we present a Python package, AnoPrimer, which exploits the Ag1000G and Af1000 datasets and allows users to rapidly design primers in An. gambiae or An. funestus, whilst summarising genetic variation in the primer binding sites and visualising the position of primer pairs. AnoPrimer allows the design of both genomic DNA and cDNA primers and hybridisation probes. By coupling this Python package with Google Colaboratory, AnoPrimer is an open and accessible platform for primer and probe design, hosted in the cloud for free. AnoPrimer is available here https://github.com/sanjaynagi/AnoPrimer and we hope it will be a useful resource for the community to design probe and primer sets that can be reliably deployed across the An. gambiae and funestus species ranges.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

Polarized M2 macrophages induced by glycosylated nano-hydroxyapatites activate bone regeneration in periodontitis therapy.

To investigate the association between periodontal macrophage polarization states and the alveolar bone levels, and to assess whether glycosylated nano-hydroxyapatites (GHANPs) could improve bone regeneration in periodontitis by inducing macrophage M2 polarization. The change of macrophage polarization state in inflammatory periodontal tissues (with bone loss) was examined using clinical gingival samples. The relationship between macrophage phenotype and bone level in periodontal bone loss and repair was evaluated using a mouse periodontitis model. The effect of GHANPs on macrophage polarization was assessed by the in vitro model of lipopolysaccharide (LPS)-stimulated inflammation. The polarization-related markers were detected by immunofluorescence staining, real-time polymerase chain reaction and enzyme-linked immunosorbent assay analysis. The therapeutic effect of GHANPs on alveolar bone loss was explored in experimental periodontitis by histological staining and micro-CT analysis. A lower macrophage M2/M1 ratio was observed in periodontitis-affected human gingival tissues. The results of animal experiments demonstrated a positive correlation between a lower Arg-1/iNOS ratio and accelerated alveolar bone loss; also, the proportion of Arg-1-positive macrophages increased during bone repair and regeneration. The administration of GHANPs partially restored M2 macrophage polarization after LPS stimulation. GHANPs increased alveolar bone repair and regeneration in experimental periodontitis induced by ligation, potentially related to their macrophage M2 transition regulation. The findings of this study indicate that the induction of macrophage M2 polarization can be considered a viable approach for enhancing inflammatory bone repair. Additionally, GHANPs show potential in the clinical treatment of periodontitis.

Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.