Sterile Solution Research Articles

Cemiplimab (Libtayo)


 CADTH reimbursement reviews are comprehensive assessments of the clinical effectiveness and cost-effectiveness, as well as patient and clinician perspectives, of a drug or drug class.
 The assessments inform non-binding recommendations that help guide the reimbursement decisions of Canada's federal, provincial, and territorial governments, with the exception of Quebec.
 This review assesses Cemiplimab (Libtayo), sterile solution for IV infusion, 50 mg/mL.
 Indication: First-line treatment of adult patients with non–small cell lung cancer (NSCLC) expressing PD-L1 (TPS ≥ 50%), as determined by a validated test, with no EGFR, ALK, or ROS1 aberrations, who have locally advanced NSCLC who are not candidates for surgical resection or definitive chemoradiation, or metastatic NSCLC.

Comparison of Oral Sirolimus, Prednisolone, and Combination of Both in Experimentally Induced Peritoneal Adhesion.

Peritoneal adhesion formation is a challenging postoperative complication. We aim to evaluate the effect of orally administered sirolimus, prednisolone, and their combination to prevent this entity. Eighty female albino underwent intraperitoneal injection of 3mL of 10% sterile talc solution to induce peritoneal adhesion, and were subsequently and randomly divided into four groups (each n=20); including a control group; 1mg/kg oral prednisolone daily in the morning; 0.1mg/kg oral sirolimus daily; and a combination group which received both drugs, with the same dosage. On the 29th day, abdominal cavities were explored, and classification was done based on Nair classification. All rats were healthy on the 29th day, in which exploration was performed. The rats in the control group had extensive intra-abdominal adhesions, while 17 (85%) rats in the control group had substantial adhesion; however, the prednisolone, sirolimus, and combination group had lesser adhesion formation. Also, 14 (70%) rats of prednisolone group, 13 (65%) of sirolimus group, and 16 (80%) of combination group had insubstantial adhesion. The decrease in the grade of peritoneal adhesion bands was highly significant in the combination group (P>0.001). The combination of sirolimus and prednisolone was effective for preventing peritoneal adhesions in rats.

Identification and quality control strategy of impurities in Zhengqing Fengtongning injection

Zhengqing Fengtongning injection is the sterile aqueous solution of Sinomenine Hydrochloride extracted from the root and stem of Sinomenium acutum, and is widely used to treat rheumatoid arthritis. Due to the processes of extraction, separation, purification, preparation and storage, some related impurities might be formed, which may cause side effects on patients. It is important to rapidly separate and identify the related impurities to ensure the safe use of Zhengqing Fengtongning injection. However, there are few literatures about the impurity in Zhengqing Fengtongning injection. In this work, ultra-high performance liquid chromatography- quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was developed to analyze impurities in both Zhengqing Fengtongning injection and its drug substance, with Sinomenine Hydrochloride as its active pharmaceutical ingredient (API). Six impurities of the Zhengqing Fengtongning injection were found. Structures of impurities 1 and 6 were confirmed by NMR and other impurities were identified from the fragmentation pattern of Sinomenine, the similarity of molecular weight and fragment ions in references. Finally, the HPLC analytical technique was developed to achieve the quantification of impurities 1 and 6. In addition, some reasonable suggestions are put forward on the quality control of Zhengqing Fengtongning injection and its drug substance based on the processes and structural characteristics of the related substances. The technical system established in this paper is helpful to strengthen the quality control of Zhengqing Fengtongning injection and improve production, and can also provide references for the production and quality control of similar drugs.

Recurrent environmental contamination in a centralized radiopharmacy unit by Achromobacter spp: Results of a large microbiological investigation

BackgroundRadiopharmaceuticals preparation unit, such as every aseptic preparation units, are strictly monitored in terms of microbiological contamination. Despite all biocontamination control procedures, our radiopharmacy unit faced repeated environmental contamination by Achromobacter spp which necessitated a large environmental investigation. MethodsMicrobiological controls were carried out using Count Tact agars (Biomérieux) for flat surfaces, dry swabbing for hard to reach areas and containers were filled with a sterile water solution (then filtrated on 0.45 µm membrane and seeded). Microbiological identification was performed by mass spectrometry (MALDI-TOF-MS, Brucker) on each positive sample. ResultsAchromobacter spp was found in 10% of the 413 samples during the 8 months investigation period. The proportion of positive samples was stable among time but their location was unpredictable. The highest inoculum was finally found in the buckets used for biocleaning. DiscussionSamples from cleaning buckets taken by dry swabbing were at first negative, but the use of a non-routinely used sampling method allowed to discover the reservoir of this persistent contamination. ConclusionThis investigation alerted us on the high microbiological risk associated with reusable plastic containers and the importance of a sampling method adapted to critical locations.

The production of Necator americanus larvae for use in experimental human infection

BackgroundAlthough there is unprecedented interest in experimental human hookworm infection, details of hookworm manufacture and characterisation have been sparsely reported. In this report, we detail the production and characterisation of Necator americanus larvae for use in a recently published clinical trial.MethodsFaeces was obtained from an experimentally infected donor. Faecal hookworm DNA was determined by quantitative PCR. Paired samples were incubated in either sterile water or sterile water mixed with antimicrobials (amphotericin and gentamicin). Coproculture was performed by modified Harada-Mori method. The harvested larvae were then processed in either sterile water or antiseptic solution. Larval yield was then calculated (larvae per gram), larval viability was determined by thermally induced motility assay and microbial burden was determined at the day of harvest, at 48 h and at 7 days.ResultsTwenty-eight faecal cultures were performed over 16 months. The faecal hookworm DNA content was variable over this time. There was no association of larval yield with faecal hookworm DNA content. Pre-treatment of faeces with antimicrobials did not influence larval yield. Larval motility was 85.3% (95% CI 79.3–91.3%). Incubation of larvae in antiseptics did not reduce viability at 14 days with a marginal mean of 68.6% (95% CI 59.1–78.1%) washed in water vs. 63.3% (95% CI 53.8 – 72.9%) when incubated in betadine (p = 0.38). Larvae washed in sterile water did not meet microbial bioburden criteria. Incubation in antiseptic resulted in acceptable microbial bioburden at 48 h but not at 7 days. Although the addition of gentamicin did reduce the microbial bio-burden acceptable levels, it was found to significantly lower larval motility at 7 days compared to incubation in sterile water and motility at 7 days 37.8% (95% CI 4.7–70.9%) vs. 67.3% (95% CI 35.2–99.3%, p < 0.001), respectively.ConclusionsDespite standardised culture methodologies and the use of a single donor, larval yield varied considerably between batches and had no association with faecal hookworm DNA. Larval viability decreases over time and the age of larvae at time of use are likely to be important. Microbial bioburden maybe temporarily reduced by incubation in antiseptics and has little effect on viability. Incubation of larvae in gentamicin is effective at reducing microbial bioburden but is deleterious to larval viability.Graphical

Morphology, Phase and Chemical Analysis of Leachate after Bioleaching Metals from Printed Circuit Boards.

The article presents the assessment of solutions and dried residues precipitated from solutions after the bioleaching process of Printed Circuit Boards (PCB) utilizing the Acidithiobacillus ferrooxidans. The obtained dried residues precipitated from bioleaching solution (leachate) and control solution were tested using morphology, phase, and chemical composition analysis, with particular emphasis on the assessment of crystalline and amorphous components. The analysis of the dried residues from leachate after bioleaching as well as those from the sterile control solution demonstrated a difference in the component oxidation—the leachate consisted of mainly amorphous spherical particles in diameter up to 200 nm, forming lacy aggregates. In the specimenform control solution larger particles (up to 500 nm) were observed with a hollow in the middle and crystalline outer part (probably Fe2O3, CuFeS2, and Cu2O). The X-ray diffraction phase analysis revealed that specimen obtained from leachate after bioleaching consisted mainly of an amorphous component and some content of Fe2O3 crystalline phase, while the dried residue from control solution showed more crystalline components. The share of the crystalline and amorphous components can be related to efficiency in dissolving metals during bioleaching. Obtained results of the investigation confirm the activity and participation of the A. ferrooxidans bacteria in the solubilization process of electro-waste components, with their visible degradation–acceleration of the reaction owing to a continuous regeneration of the leaching medium. The performed investigations allowed to characterize the specimen from leachate and showed that the application of complementary cross-check of the micro (SEM and S/TEM) and macro (ICP-OES and XRD) methods are of immense use for complete guidance assessment and obtained valuable data for the next stages of PCBs recycling.

First Report of Pectobacterium punjabense Causing Blackleg and Soft Rot on Potato in Hebei and Fujian Province, China.

Pectobacterium spp. and Dickeya spp. cause blackleg and soft rot on potato worldwide (Charkowski, 2018). Potato plants (cv. Favorita or Jizhang 8#) with blackleg symptoms (vascular browning of crown stems, Fig. S1) were observed in the field in Zhangjiakou, Hebei province in 2018, and in Ningde, Fujian Province in 2019, in China. The disease incidence was around 50% and 10% in Zhangjiakou (5 ha) and Ningde (4 ha), respectively. Diseased plants (3 from each site) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, 4 bacterial colonies in total which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Two colony sequences that showed more than 99% sequence identity to Pectobacterium punjabense type strain SS95 (MH249622) were submitted to the GenBank ( accession numbers: OK510280, MT242589). Additionally, six housekeeping genes proA (OK546205, OK546199), gyrA (OK546206, OK546200), icdA (OK546207, OK546201), mdh (OK546208, OK546202), gapA (OK546209, OK546203), and rpoS (OK546210, OK546204) of these two isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with MH249622T . Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 2 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch's postulate was performed on potato seedlings and potato tubers (cv. Favorita) by injecting 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems or the tubers and kept at 100% humidity and 21°C for 1 day. Four days after inoculation, the infected area of the inoculated seedlings rotten and turned black, while the controls were symptomless (Fig. S4). Two days after inoculation, the infected tubers rotten and turned black, while the controls were symptomless (Fig. S4). Bacterial colonies were reisolated from these symptomatic tissues and identified using the same methods described above. Blackleg on potato plants or soft rot on potato has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium parmentieri, Pectobacterium polaris in China (Zhao et al., 2018; Cao et al., 2021; Wang et al., 2021). To our knowledge, this is the first report of blackleg/soft rot of potato caused by Pectobacterium punjabense in China. We believe that this report will draw attention to the management of this pathogen in China.

BIOSYNTHESIS OF PHYTOHORMONES AND VIRULENCE OF АZOTOBACTER CHROOCOCCUM 2.1 — CUCUMIS SATIVUS L. INOCULANT

Objective. To study the properties (production of phytohormonal substances and virulence) of a new highly effective strain of associative bacteria of the genus Azotobacter. Methods. Phytohormonal activity was established by bioassays. Microbiological (obtaining a suspension of Azotobacter chroococcum 2.1, determining the titre by sowing on a digest medium; microscopy of stained imprint smears of the internal organs of the studied animals and cultivation of tissue samples on a digest medium) and patho-anatomical (determination of possible invasiveness and dissemination of bacteria in the tissue of internal organs of animals) methods were applied. The study was conducted on a model of outbred white mice, after oral dosing of a suspension of live bacterial cells ranged from 0.5×109 to 5×109 CFU in 0.5 cm3 of sterile isotonic sodium chloride solution per mouse and intraperitoneal dosing ranged from 1×109 to 5×109 CFU/0.5 cm3. Statistical calculations were carried out using analysis-of-variance method. Results. A new A. chroococcum 2.1 strain synthesizes auxin-like substances more intensively than gibberellin-like and cytokinin-like substances. A 1:100 dilution of the culture fluid is the most effective. The obtained results of the studies on the possible pathogenicity of bacteria indicate that no deviations in the behaviour of mice were detected after oral dosing of a suspension of living Azotobacter cells. Microbiological tests of the internal organs of the experimental animals after the beginning of the tests and after 15 days revealed that Azotobacter is not infective, does not reproduce and does not disseminate in the body of warm-blooded animals. Intraperitoneal and oral dosing of a live cell culture suspension of A. chroococcum 2.1 did not cause bacterial invasion into the internal organs of white mice. Retrocultures were not detected. Conclusion. A. chroococcum 2.1 produces auxins, gibberellins and cytokinins. The parabolic nature of the influence of dilutions of the culture liquid of the studied strain A. chroococcum 2.1 on the test cultures (wheat coleoptiles, corn mesocotyles and cucumber cotyledons) was revealed, which is a consequence of the production of phytohormonal substances. The new strain produces auxin-like substances most intensely. The obtained data indicate the avirulence of the strain for warm-blooded animals (LD50 intraperitoneal &gt; 5 billion cells/mouse, LD50 oral &gt; 5 billion cells/mouse). According to the data on the lack of virulence, without taking into account the levels of toxigenicity, toxicity, allergenic capacity and dysbiotic action, A. chroococcum 2.1 can be considered non-pathogenic.

Functional activity of peripheral blood mononuclear cells in patients with non-Hodgkin's lymphomas receiving treatment after COVID-19 disease.

e19564 Background: Patients with non-Hodgkin's lymphomas (NHL) develop abnormalities in the structural and functional organization of the immune system leading to immune deficiency. Chemotherapy (CT) in patients after SARS-CoV infection is associated with a more severe disease course affecting the treatment results. The cytokine-producing activity (CPA) of blood cells is poorly studied, while it determines the effectiveness of antitumor and anti-infective functions of the immune system. The purpose of this study was to evaluate CPA of peripheral blood mononuclear cells in patients receiving treatment for NHL after COVID-19. Methods: The study included 8 patients with large B-cell NHL with PCR-confirmed COVID-19 infection in past medical history. All patients received from 3 to 4 chemotherapy cycles. K2EDTA blood samples obtained before and after 3-4 CT cycles were divided into 2 parts after dilution with a sterile nutrient medium solution: part 1, to assess spontaneous CPA; part 2, with addition of a sterile mitogen (phytohemagglutinin 4 μg, concanavalin A 4 μg, and lipopolysaccharide 2 μg) to assess stimulated CPA. The samples were incubated for 24 hours at 370C, and the levels of IL-1β, IL-6, IL-8, IL-10, IL-18, IL-4, IL-2, TNF- α, INF-ɣ, INF-α were determined in the obtained plasma. The stimulation coefficient (SC) was calculated as the ratio of stimulated CPA to spontaneous CPA. Results: 3-4 CT cycles in patients after COVID-19 was accompanied by an elevation of spontaneous CPA of the blood cells IL-6, INF-ɣ, TNF-α, IL-8, compared to the initial levels, by 678%, 127%, 64% and 57%, respectively. The ability of cells to spontaneous production of IL-10 and INF-α decreased by 30% and 100%. The mitogen-induced CPA of mononuclear cells in relation to IL-10, IL-6, IL-2, IL-1β and INF-α increased by 300%, 130%, 92%, 52% and 52%, respectively. Stimulated CPA in relation to INF-ɣ decreased by 21% compared to initial levels. As a result of the revealed CPA changes, SC in NHL patients after COVID-19 receiving CT increased, compared to the initial levels, by 465%, 92% and 48% respectively for IL-10, IL-2, IL-1β, as well as the appearing ability to INF-α production. SC for IL-6, INF-ɣ, TNF-α, and IL-8 decreased by 70%, 66%, 33% and 27% respectively. Conclusions: Certain features of spontaneous and mitogen-activated CPA of blood mononuclear cells were revealed in NHL patients after COVID-19, indicating a change in the functional activity of immune cells which could affect the development of the disease and the effectiveness of the therapy. The data obtained require additional studies and can be used to assess the condition of patients, as well as to predict the therapy efficacy.

A validated UPLC-MS/MS method for quantification of glutamine in plasma and pharmacokinetic study of oral glutamine tablets in healthy Beagles.

This study aimed to clarify the laws of glutamine tablets absorption, distribution, and metabolism in Beagles and to provide a basis for formulating dosing regimens. Twelve healthy Beagles were enrolled the absolute bioavailability study with a crossover design . Glutamine tablets (240 mg/kg b.w.) or glutamine sterile solution (60 mg/kg b.w.) were administered. A method for the determination of glutamine in Beagles' plasma by UPLC-MS/MS was established, with high sensitivity, specificity, and simplicity. Based on the study of endogenous glutamine concentration, the mean concentration of the four time points before drug administration was selected as the background concentration of glutamine. Pharmacokinetic parameters were calculated by non-compartment model. The Cmax of glutamine was 136.11 ± 72.51 μg/ml, Tmax was 0.85 ± 0.29 h, and t1/2λz was 0.42 ± 0.27 h after oral administration. The AUC0-t of glutamine was 116.30 ± 75.15 h·μg/ml vs. 44.55 ± 22.48 h·μg/ml following oral and IV administration, respectively, with an absolute bioavailability of 64.74% ± 19.18%. The results showed glutamine was quickly absorbed and eliminated in Beagles with high bioavailability. Therefore, glutamine is suitable to be prepared as oral tablets and recommended to shorten the dosing interval.

Sentinel lymph node mapping for endometrial cancer: Opportunity for medical waste reform

ObjectiveAs healthcare expenditures continue to rise, identifying mechanisms to reduce unnecessary costs is critical. The objective of this study is to estimate the annual cost of wasted indocyanine green (ICG) used for sentinel lymph node mapping in patients with endometrial cancer. MethodsUsing the Surveillance, Epidemiology, and End Results program database and Premier database, we determined the annual number of cases in which sentinel lymph node mapping with ICG would be used and the median cost of ICG to institutions and patients, respectively. We assumed that gynecologic oncologists use 2-4 mL (20–40%) of the currently available ICG vial kit (25 mg per 10 mL) per case. Estimated waste was then calculated using cost as a measure of institutional waste and charge as excess cost transferred to patients or payers. ResultsAn estimated 45,864 cases of localized endometrial cancer were identified and eligible for sentinel lymph node (SLN) mapping. The mean total cost associated with ICG was 99.20 and the mean charge was $483.64. The estimated excess annual cost to hospitals was $2,729,825 to $3,639,767. Similarly, using mean charge data, the annual cost of wasted drug for patients and payers was $13,308,999 to $17,745,332. ConclusionsThe annual cost of wasted ICG due to its current manufactured vial size exceeds $2 million for hospitals and $13.3–$17.7 million for patients. We suggest ICG vials should be packaged in a 10 mg vial kit (2–4 mL sterile solution) to avoid drug waste and the financial impact to institutions and patients.

Exposure-Response Modeling and Simulation to Support Human Dosing of Botulism Antitoxin Heptavalent Product.

Botulism antitoxin heptavalent (A, B, C, D, E, F, and G – Equine; BAT) product is a sterile solution of F(ab')2 and F(ab')2‐related antibody fragments prepared from plasma obtained from horses that have been immunized with a specific serotype of botulinum toxoid and toxin. BAT product is indicated for the treatment of symptomatic botulism following documented or suspected exposure to botulinum neurotoxin serotypes A to G in adults and pediatric patients. Pharmacokinetic and exposure‐response models were used to explore the relationship between BAT product exposure and the probability of survival, and the occurrence of relevant moderate clinical signs observed during the preclinical development of BAT product to justify the clinical dose. The predicted probability of survival in humans for all serotypes of botulinum neurotoxin was more than 95.9% following intravenous administration of one vial of BAT product. Furthermore, this BAT product dose is expected to result in significant protection against clinical signs in human adults for all botulinum neurotoxin serotypes. Our exposure response model indicates that we have sufficient antitoxin levels to give full protection at various theoretical exposure levels and, based on neutralization capacity/potency of one dose of BAT product, it is expected to exceed the amount of circulating botulinum neurotoxin.

Evaluation of a single-shot of a high-density viscoelastic solution of hyaluronic acid in patients with symptomatic primary knee osteoarthritis: the no-dolor study

BackgroundPronolis®HD mono 2.5% is a novel, one-shot, high-density sterile viscoelastic solution, recently available in Spain, which contains a high amount of intermediate molecular weight hyaluronic acid (HA), highly concentrated (120 mg in 4.8 mL solution: 2.5%). The objective of the study was to analyze the efficacy and safety of this treatment in symptomatic primary knee osteoarthritis (OA).MethodsThis observational, prospective, multicenter, single-cohort study involved 166 patients with knee OA treated with a single-shot of Pronolis®HD mono 2.5% and followed up as many as 24 weeks.ResultsCompared with baseline, the score of the Western Ontario and McMaster Universities Arthritis Osteoarthritis Index (WOMAC) pain subscale reduced at the 12-week visit (primary endpoint, median: 9 interquartile range [IQR]: 7–11 versus median: 4; IQR: 2–6; p < 0.001). The percentage of patients achieving > 50% improvement in the pain subscale increased progressively from 37.9% (at 2 weeks) to 66.0% (at 24 weeks). Similarly, WOMAC scores for pain on walking, stiffness subscale, and functional capacity subscale showed significant reductions at the 12-week visit which were maintained up to the 24-week visit. The EuroQol visual analog scale score increased after 12 weeks (median: 60 versus 70). The need for rescue medication (analgesics/nonsteroidal anti-inflammatory drugs) also decreased in all post-injection visits. Three patients (1.6%) reported local adverse events (joint swelling) of mild intensity.ConclusionsIn conclusion, a single intra-articular injection of the high-density viscoelastic gel of HA was associated with pain reduction and relief of other symptoms in patients with knee OA.Trial registrationClinicalTrial# NCT04196764

Co-administration of recombinant chicken IL-7 or chicken NK-lysin peptide 2 increases efficacy of Eimeria tenella elongation factor-1α in Eimeria maxima infected chicken

Abstract This study was conducted to improve the efficacy of recombinant Eimeria tenella elongation factor-1α (EF-1α) antigen by co-administrating recombinant chicken IL-7 or chicken NK-lysin peptide 2 in Eimeria maxima-challenged chickens. On day 4, birds were administered with following five groups: (CON) control birds without Eimeria infection, (NC) sterile PBS solution plus adjuvant (ISA 78 VG), (COM 1) 100 μg of recombinant EF-1α plus ISA 78 VG, (COM 2) COM 1 component plus 1 μg of recombinant chIL-7, and (COM 3) COM 2 component plus 5 μg of cNK2 peptide. One week after, the boosting immunization were administrated with same concentration of components as the primary immunization. All birds except CON were orally inoculated with E. maxima (1.0 × 104 oocysts/bird) on day 19. Animal experiment demonstrated that the birds immunized with recombinant EF-1α showed greater serum anti EF-1α level, and co-administration of recombinant chIL-7 increased the level of IL-7 in serum. Vaccination of COM 2 mitigated intestinal lesion formation at 6 days post-infection (dpi) and increased body weight at 6 dpi and 9 dpi. Vaccination of COM 3 reduced fecal oocyst shedding compared with NC. E. maxima infection up-regulated expression level of jejunal proinflammatory cytokines (IL-β, IL-17F) and type I cytokines (IFN-γ and IL-10), and those expression levels were decreased by administration of COM 1, COM 2 or COM 3. Down-regulated expression of tight junction (TJ) protein (Jam2 and Occludin) by E. maxima challenge were increased by COM 2or COM 3, respectively. Overall animal experiment results demonstrated a potential effect that co-administration of recombinant IL-7 or cNK-2 peptide can increase the efficacy of recombinant EF-1α in E. maxima-infected chicken. Funding was partly supported by ARS CRIS 8042-32000-107-00D, NIFA SBIR 2015-33610-23499, and NIFA SBIR 2016-33610-25686.

Anti-PCSK9 monoclonal antibody attenuates high-fat diet and zymosan-induced vascular inflammation in C57BL/6 mice by modulating TLR2/NF-ƙB signaling pathway.

Objective(s):Excess intake of a high-fatty diet (HFD) together with zymosan administration mediates vasculitis response which leads to impaired serum lipid levels and causes arterial stiffness. In the development of new cholesterol-lowering medications, PCSK9 inhibitor (proprotein convertase subtilisin/kexin type 9) is an emerging therapeutic. The goal of the present study was to see whether anti-PCSK9 mAb1 might prevent vasculitis in C57BL/6 mice by blocking TLR2/NF-B activation in HFD and Zymosan-induced vasculitis. Materials and Methods:Protein-protein molecular docking was performed to validate the binding affinity of anti-PCSK9 mAb1 against TLR2. Under the experimental study, mice were randomly allocated to the following groups: Group I: standard mice diet (30 days) + Zymosan vehicle (sterile PBS solution of 5mg/ml on 8th day); Group II: HFD (30 days) + Zymosan ( single IP dose 80 mg/kg on day 8th); Group III: HFD+Zymosan + anti-PCSK9 mAb1 (6 mg/kg, s.c. on 10th and 20th days); Group IV: HFD+Zymosan+anti-PCSK9 mAb1 (10 mg/kg, s.c. on 10th and 20th days).Results:In comparison with the low dose of anti-PCSK9 mAb1 (6 mg/kg), the high dose of anti-PCSK9 mAb1 (10 mg/kg) together with HFD and Zymosan inhibited vasculitis more effectively by decreasing aortic TLR2 and NF-B levels, reducing serum TNF- and IL-6, and up-regulating liver LDLR levels, which down-regulated serum LDL-C and improved serum lipids levels. Histopathological studies showed that anti-PCSK9 mAb1 treatment reduced plaque accumulation in the aorta of mice.Conclusion:These findings indicate that anti-PCSK9 mAb1 has therapeutic potential in reducing HFD and Zymosan-induced vascular inflammation.

Retrieving the in vivo Scopoletin Fluorescence Excitation Band Allows the Non-invasive Investigation of the Plant-Pathogen Early Events in Tobacco Leaves.

In this study, we developed and applied a new spectroscopic fluorescence method for the in vivo detection of the early events in the interaction between tobacco (Nicotiana tabacum L.) plants and pathogenic bacteria. The leaf disks were infiltrated with a bacterial suspension in sterile physiological solution (SPS), or with SPS alone as control. The virulent Pseudomonas syringae pv. tabaci strain ATCC 11528, its non-pathogenic ΔhrpA mutant, and the avirulent P. syringae pv. tomato strain DC3000 were used. At different post-infiltration time–points, the in vivo fluorescence spectra on leaf disks were acquired by a fiber bundle-spectrofluorimeter. The excitation spectra of the leaf blue emission at 460 nm, which is mainly due to the accumulation of coumarins following a bacterial infiltration, were processed by using a two-bands Gaussian fitting that enabled us to isolate the scopoletin (SCT) contribution. The pH-dependent fluorescence of SCT and scopolin (SCL), as determined by in vitro data and their intracellular localization, as determined by confocal microscopy, suggested the use of the longer wavelength excitation band at 385 nm of 460 nm emission (F385_460) to follow the metabolic evolution of SCT during the plant–bacteria interaction. It was found to be directly correlated (R2 = 0.84) to the leaf SCT content, but not to that of SCL, determined by HPLC analysis. The technique applied to the time-course monitoring of the bacteria–plant interaction clearly showed that the amount and the timing of SCT accumulation, estimated by F385_460, was correlated with the resistance to the pathogen. As expected, this host defense response was delayed after P. syringae pv. tabaci ATCC 11528 infiltration, in comparison to P. syringae pv. tomato DC3000. Furthermore, no significant increase of F385_460 (SCT) was observed when using the non-pathogenic ΔhrpA mutant of P. syringae pv. tabaci ATCC 11528, which lacks a functional Type Three Secretion System (TTSS). Our study showed the reliability of the developed fluorimetric method for a rapid and non-invasive monitoring of bacteria-induced first events related to the metabolite-based defense response in tobacco leaves. This technique could allow a fast selection of pathogen-resistant cultivars, as well as the on-site early diagnosis of tobacco plant diseases by using suitable fluorescence sensors.

Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An In Vitro Study.

The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files via Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Fifty-two k-files of size #25 and length 21 mm were taken for the study. All the 52 files were presterilized in an endodontic instrument box via autoclave. Bacillus stearothermophilus spore suspension was prepared and all the presterilized files were contaminated with stearothermophilus spore suspension in a sterile Petri dish under vacuum hood safety. Later, the test files were randomly divided into four groups of 13 each and subjected to four different methods of sterilization- Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Files were then be placed in thioglycollate media containing test tubes and incubated in an incubator at 55°C and checked for turbidity at 3 days and 21 days. The result revealed that there was no Turbidity present in test tubes on both the 3rd and 21st day for autoclave. In all the remaining sterilization procedures there was some amount of turbidity present. In terms of sterilization provided autoclave provides complete sterilization and glutaraldehyde solution is the least effective.The specificity of Bacillus stearothermophilus was then confirmed with a sugar test viz., starch hydrolysis test which gave a positive result confirming the presence of Bacillus stearothermophilus. We can conclude that autoclave is the perfect process of sterilization providing 100% sterility and although Glass-bead didn't provide 100% sterility, it can be used as an alternative if autoclave is not available. Ameer B, Khatib MS, Peerzade SM, et al. Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An In Vitro Study. Int J Clin Pediatr Dent 2022;15(2):149-152.

Enhanced presence of serotonin in nasal cavity after autologous stimulation.

Serotonin, which is a vasoactive amine, is an important neurotransmitter and is involved in many behavioral and psychological phenomena, such as pain, appetite, mood, and sleep. The primary purpose of our study was to investigate the effect of high-pressure administration of sterile physiological saline isotonic solution (HpPSIS) into nasal cavity and to determine the expression of the serotonin. The study was made in two branches, the previous with 14 volunteers, the subsequent study with 40 patients with mild anxiety disorder. The middle third of the inferior turbinate epithelial cells on the right nostril was scraped using a sterile curette and indicated as (pre), then, a spray of sterilized isotonic solution at high pressure on the left nostril was delivered, and 5 minutes later a similar stimulation was delivered on the same nostril. The stimulation was made with a specific spray dispenser. The middle third of the inferior turbinate epithelial cells on the left nostril was scraped using a sterile curette and indicated as (post). Then, based on the first part of our study, we started the second part and gave a treatment on forty new patients with anxiety disorder. The results of these studies highlight the possibility of endogenous enhancement of serotonin by stimulation of mast cells. In the first part of the study, Serotonin significantly increased in protein extracts after treatment (64.35±5.33 vs. 10.97±2.17; unpaired two tailed t-test, t=9.8, df=24, p≤0.0001; F=6.035; DFn=12; DFd=12). In the second part of the study, in patients treated with HpPSIS, we observed improvement of mood, after one, two and three months, with a statistically significant reduction of DASS-21, while no reduction was observed in control patients, treated with normal pressure commercial spray. This pilot study showed that the topical treatment of HpPHIS increases serotonin levels in nasal cavity. The observation reported in this study opens the way to a new valid strategy to enhance the level of endogenous serotonin. We observed a significant improvement of ASI on patients during HpPHIS therapy.

СОЧЕТАННОЕ ПРИМЕНЕНИЕ ХИРУРГИЧЕСКОГО ИССЕЧЕНИЯ ЛУЧЕВОЙ ЯЗВЫ И ТРАНСПЛАНТАЦИИ СИНГЕННЫХ КЛЕТОК СТРОМАЛЬНО-ВАСКУЛЯРНОЙ ФРАКЦИИ ЖИРОВОЙ ТКАНИ ПРИ ЛЕЧЕНИИ ТЯЖЕЛЫХ МЕСТНЫХ РАДИАЦИОННЫХ ПОРАЖЕНИЙ В ЭКСПЕРИМЕНТЕ

Purpose: To study the therapeutic efficacy of the combined use of surgical excision of radiation ulcers and transplantation of syngeneic cells of the stromal-vascular fraction (SVF) of adipose tissue in the treatment of severe local radiation lesions. Material and methods: Experiments were conducted on rats males of the inbred line Wista‒Kyoto weighing 230‒250 g. Animals were irradiated on an X-ray unit locally in the iliac-lumbar region of the back at a dose of 110 Gy (voltage on the tube 30 kV, current strength 6.1 mA, filter 0.1 mm Al), at a dose rate of 20,0 Gy/min. Irradiation field area 8.2 cm2. Radiation exposure made it possible to obtain severe skin lesions in rats with long-term non-healing ulcers, and without critical radiation exposure to the underlying tissues. Morphological study showed that the area of skin necrosis (with a maximum in the center of the irradiation zone) was formed by 21‒23 days after irradiation and was characterized by the presence of pronounced changes in all layers of the skin. Surgical excision of radiation ulcers was performed on 22 days after exposure to radiation. The affected tissue was removed to the entire depth of the skin up to the fascia of skeletal muscles. Excision of the skin was carried out, retreating 6‒8 mm from the outer border of the radiation ulcer. Cell transplantation was carried out twice 5 and 12 days after excision of the ulcer. SVF cells were obtained from intact animals from subcutaneous adipose tissue treated with trypsin. A suspension of SVF cells of adipose tissue in 1 ml of a sterile Solution of Henks was injected under the skin at 5 points around the excision zone, retreating 5‒7 mm from the edge of the focus. The number of injected cells in one transplant was 2.5×106. Results: The area of radiation ulcers in rats of the control group in the period from 27 to 105 days after irradiation slowly decreased from 2.8±0.2 cm2 to 1.2 ± 0.2 cm2. Excision of radiation ulcers led to rapid complete visible healing of the wound defect (already by 70 days after irradiation) with the formation of atrophic scars. Transplantation of SVF syngeneic cells increased the rate of healing of the surgical wound. Conclusion: The results show that surgical excision of radiation ulcers in combination with cell therapy may be an effective way to treat severe local radiation lesions.

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.