Chitosan nanoparticles as a new technique in gene transformation into different plants tissues

The utilization of chitosan nanoparticles is a novel technique for gene transformation into plant tissues. It takes a few minutes to transform gene to plant. UidA gene was detected in <i>Escherichia coli</i> (K12 strain) using polymerase chain reaction analysis by UidA-specific primers. The gene was transformed into the explants of two different plant species (<i>Solanum tuberosum</i> and <i>Paulownia tomentosa</i>). These plants have different natures as crop and woody plants respectively. Therefore, they have different abilities to express the UidA gene. The gene is expressed into blue color in plant tissues due to the formation and expression of the GUS enzyme. The transformation of the UidA gene was detected morphologically by the formation of blue color; and molecular using PCR. Chitosan nanoparticles were characterized by UV/Visible spectroscope and photographing with a transmission electron microscope (TEM). As a result of this research, it is suggested that chitosan nanoparticles be used in gene transformation into plant tissues. Because it is safe, quick, and inexpensive, as well as biocompatible and biodegradable.

Orphan Receptor GPR50 Improves Inflammation and Insulin Signaling in 3T3-L1 Preadipocyte

Purpose: The goal of this study was to investigate the effect of orphan G Protein-Coupled Receptor 50 (GPR50) receptor on inflammation and insulin signaling in 3T3-L1 preadipocyte.Subjects and Methods: A high-fat diet (HFD)-induced obesity-T2DM (Type 2 Diabetes Mellitus) mouse model was used in this research, and high expression of GPR50 in mouse adipose tissue was screened by microarray technology. Expression of GPR50 in 3T3-L1 cell line and obesity-T2DM mouse adipose tissue was confirmed. To gain more insight into the potential role of this new target in obesity-associated IR development, a GPR50 knockout cell line was constructed in 3T3-L1 cell line. Inflammatory cytokine levels and insulin signaling pathways in the GPR50 knockout 3T3-L1 cell line were determined by quantitative real-time polymerase chain reaction analysis and western blot.Results: GPR50 expression was significantly increased in adipose tissue of obesity-T2DM mice. GPR50 deficiency increased inflammation in 3T3-L1 cells. In addition, GPR50 deficiency induced the phosphorylation of AKT and insulin receptor substrate (IRS)1. Furthermore, GPR50 knockout 3T3-L1 cell line had suppressed PPAR-γ expression.Conclusions: These data demonstrated a novel target GPR50 can affect inflammation and insulin signaling in adipocytes. Furthermore, the effects are mediated through the regulation of insulin signaling and PPAR-γ expression.

CircKIF4A Enhances Osteosarcoma Proliferation And Metastasis By Sponging MiR-515-5p And Upregulating SLC7A11

Background Circular RNAs (circRNAs) are forms of non-coding RNAs that have crucial roles in regulation of various biological processes of several malignant tumors. circKIF4A is closely associated with malignant progression of a variety of cancers. However, the molecular mechanisms as well as roles of circKIF4A in osteosarcoma (OS) have not yet been clearly elucidated. Methods We evaluated the expression of circKIF4A in OS. Colony-formation, cell counting kit-8 (CCK-8), transwell and mice metastasis model assays were done to explore the roles of circKIF4A in vitro and in vivo. TargetScan database, double luciferase, quantitative reverse transcription polymerase chain reaction analysis (RT-qPCR), and RNA immunoprecipitation (RIP) were done to investigate the associated molecular mechanisms. Results In both OS cells and tissues, circKIF4A (hsa_circ_0007255) was found to be upregulated. In vitro and in vivo, circKIF4A knockdown markedly suppressed OS proliferation as well as metastasis. circKIF4A enhanced OS growth as well as metastasis by sponging miR-515-5p and by upregulating SLC7A11. Conclusions We identified the biological significance of the circKIF4A-miR-515-5p-SLC7A11 axis in OS cell proliferation and metastasis, which is important in OS monitoring and treatment. More studies on circKIF4A will inform on the diagnostic markers for early OS screening.

Intraventricular tuberculosis abscess in an immunocompromised patient: clinical vignette

Tuberculosis is caused by Mycobacterium tuberculosis. Tuberculosis of the central nervous system is common and manifestations include meningeal and intraparenchymal diseases. However, intraventricular tuberculous abscess is a rare manifestation of intracranial tuberculous infection. We present a case of an immunocompromised female patient with high-grade fever and signs of meningism. The computed tomography and magnetic resonance imaging (MRI) of the brain showed hydrocephalus with rim-enhancing lesion in the right lateral ventricle. The MRI demonstrated a hypointense signal on T1-weighted imaging, hyperintense signal on T2-weighted imaging, and mild restricted diffusion in diffusion-weighted imaging. She underwent emergency external ventricular drainage and frank pus was drained. Diagnosis of tuberculosis was made via polymerase chain reaction analysis and culture. Understanding the intracranial manifestation of neurotuberculosis is imperative to arrive at the diagnosis correctly and ensure prompt treatment.

Optimized QRT-PCR Approach for the Measurable Impact of Adjuvant Cholecalciferol Therapy in Ameliorating Cytokine Gene Expression

The endemic Vitamin D deficiency in Pakistan and the current COVID-19 epidemic have converged into a double whammy scenario in Pakistan [1]. Nutritional epigenomic studies have highlighted Vitamin D as a master Vitamin influencing various genomic expressions through its active metabolite 1α,25-dihydroxyvitamin D3 [2]. The objective of this study was to evaluate the measurable impact of adjuvant Cholecalciferol therapy in the Cytokine gene expression of COVID-19 patients by quantitative Real-Time Polymerase Chain Reaction analysis. The trial was a randomized control prospective open label interventional trial done on moderate to severe COVID-19 patients with deranged inflammatory and coagulation biomarkers. SunnyD STAT (Vitamin D3 200000 IU) softgels were given at Day 1, Day 3 and Day 5 of the treatment. Optimized quantitative Real-Time Polymerase Chain Reaction analysis showed decreased genetic expressions of Interleukin 6 (IL-6), Interleukin 2RA (IL-2RA) and Tumor Necrosis Factor (TNF-a) in the interventional group against the age and co-morbidities matched controls, providing molecular and genetic level evidence for the purported mechanism of amelioration of Cytokines induced pathogenic inflammation. However, inherent limitations of the design restrict the generalizability of the results and warrants caution for extrapolation. We recommend randomized placebo-controlled trials with larger sampling and genome wide profiling to infer more definite interpretations.

Validation of SFRP1 Promoter Hypermethylation in Plasma as a Prognostic Marker for Survival and Gemcitabine Effectiveness in Patients with Stage IV Pancreatic Adenocarcinoma

No reliable predictive blood-based biomarkers are available for determining survival from pancreatic adenocarcinoma (PDAC). This combined discovery and validation study examines promoter hypermethylation (ph) of secreted frizzled-related protein 1 (SFRP1) in plasma-derived cell-free DNA as an independent prognostic marker for survival and Gemcitabine effectiveness in patients with stage IV PDAC. We conducted methylation-specific polymerase chain reaction analysis of the promoter region of the SFRP1 gene, based on bisulfite treatment. Survival was analyzed with Kaplan–Meier curves, log-rank test, and Cox regression. The discovery cohort included 40 patients, 25 receiving Gem. Gem-treated patients with phSFRP1 had a shorter median overall survival (mOS) (4.4 months) than unmethylated patients (11.6 months). Adjusted Cox-regression yielded a hazard rate (HR) of 3.48 (1.39–8.70). The validation cohort included 58 Gem-treated patients. Patients with phSFRP1 had a shorter mOS (3.2 months) than unmethylated patients (6.3 months). Adjusted Cox regression yielded an HR of 3.53 (1.85–6.74). In both cohorts, phSFRP1 was associated with poorer survival in Gem-treated patients. This may indicate that tumors with phSFRP1 are more aggressive and less sensitive to Gem treatment. This knowledge may facilitate tailored treatment of patients with stage IV PDAC. Further studies are planned to examine phSFRP1 in more intensive chemotherapy regimens.

Rapid growth atypical mycobacteria infection associated with growth hormone injections: a case report

Introduction. Infections caused by fast growing mycobacteria have increased markedly worldwide. They are normally associated with trauma, surgery or cosmetic interventions. Paraguay has a deficit in sanitary control including clinics, private practices, and aesthetic centres. This situation is accompanied by the easy access to drugs, which leads to the performance of exclusively medical aesthetic procedures by people without professional knowledge or training. Case report. A 26-year-old female patient comes to a medical consultation with pain and bruising in the abdominal area with more than 3 months of progression, without fever or apparent cause. Later, she confessed to the application of subcutaneous injections of ‘growth hormones’ at the gym. Excisional biopsy of the lesions was carried out for anatomopathological and microbiological studies. In addition, the use of polymerase chain reaction analysis was indicated because of the strong suspicion of an atypical mycobacterial infection. The Ziehl-Neelsen staining was negative for BAAR, and the PAS-Hematoxylin negative for fungal elements. When performing the culture, the growth of atypical mycobacteria was observed on chocolate and blood agar medium culture. Through the polymerase chain reaction study, it was possible to identify the atypical mycobacterium as ‘Mycobacterium abscessus’. Conclusion. The irresponsible application of medications by people without professional authorization or biosafety precautions can lead to the development atypical infections that are difficult to diagnose and treat. This situation could lead to serious complications and even death.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

Antimicrobial Activity of Ca-Alginate/Chitosan Nanocomposite Loaded with Camptothecin

The main aim of this study was to prepare antimicrobial nanocomposites consisting of alginate, chitosan, and camptothecin (CPT). CPT-loaded calcium alginate (Ca-Alg2) and calcium alginate/chitosan (Ca-Alg2-CH) nanomaterials were synthesized and characterized using infrared (IR) spectroscopy, X-ray diffraction (XRD), UV-Vis spectroscopy, and scanning electron microscopy (SEM). The antimicrobial activity and the genetic effects of Ca-Alg2/CPT and Ca-Alg2-CH/CPT nanomaterials on Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia were studied. The repetitive element polymerase chain reaction analysis technique was used to assess the changes in the bacterial genetic material due to the processing of the nanomaterials. The results showed the presence of a strong chemical interaction between alginate and chitosan, and CPT was loaded successfully in both Ca-Alg2/CPT and Ca-Alg2-CH/CPT nanomaterials. Furthermore, the antimicrobial test showed that the Ca-Alg2/CPT nanocomposite was susceptible to S. aureus, E. coli, and K. pneumonia; on the other hand, Ca-Alg2-CH/CPT nanocomposite was more susceptible to E. coli and K. pneumonia and was resistant to S. aureus. The results showed that the Ca-Alg2/CPT nanocomposite was less efficient than Ca-Alg2-CH/CPT nanocomposite in killing Gram-negative treated bacteria. Moreover, results revealed that the PCR analysis revealed a polymorphic banding pattern. This observation provides an excellent guide to the ability of some polymers to induce point mutations in DNA.

Identification and Expression Profiling of Circulating MicroRNAs in Serum of Cysticercus pisiformis-Infected Rabbits

Cysticercus pisiformis (C. pisiformis), the larval form of Taenia pisiformis, parasitize mainly the liver, omentum and mesentery of rabbits and cause huge economic losses in the rabbit breeding industry. MicroRNA (miRNA), a short non-coding RNA, is widely and stably distributed in the plasma and serum. Numerous data demonstrates that, after parasitic infection, miRNAs become the key regulatory factor for controlling host biological processes. However, the roles of serum miRNAs in C. Pisiformis-infected rabbits have not been elucidated. In this study, we compared miRNA expression profiles between the C. pisiformis-infected and healthy rabbit serum using RNA-seq. A total of 192 miRNAs were differentially expressed (fold change ≥ 2 and P < 0.05), including 79 up- and 113 downregulated miRNAs. These data were verified by qRT-PCR (real time quantitative polymerase chain reaction) analysis. Additionally, GO analysis showed that the target genes of these dysregulated miRNAs were most enriched in cellular, single-organism and metabolic processes. KEGG pathway analysis showed that these miRNAs target genes were involved in PI3K-Akt, viral carcinogenesis and B cell receptor signaling pathways. Interestingly, after aligning clean reads to the T. pisiformis genome, four (miR-124-3p_3, miR-124-3p_4, miR-124a and novel-miR1) T. pisiformis-derived miRNAs were found. Of these, novel-miR1was upregulated in different periods after C. pisiformis infection, which was verified qRT-PCR, and pre- novel-miR-1 was amplified from the cysticerci by RT-PCR, implying novel-miR-1 was derived from C. pisiformis and has great potential for the diagnosis of Cysticercosis pisiformis infection. This is the first investigation of miRNA expression profile and function in the serum of rabbits infected by C. pisiformis, providing fundamental data for developing diagnostic targets for Cysticercosis pisiformis.