Three-step Protocol Research Articles

Highly Regioselective Protecting-Group-Free Synthesis of the Antimalarial Drug MMV693183.

MMV693183 is a promising antimalarial drug candidate that works for uncomplicated malaria treatment and resistance management. Herein, we report an efficient and highly regioselective synthesis of MMV693183. This novel synthetic method highlights a three-step route with an overall yield of 46% from readily available starting materials. The key to the success lies in (1) utilizing the subtle difference of the two amino groups in the starting material (S)-propane-1,2-diamine dihydrochloride without amino protection and (2) identifying the L-(+)-tartaric acid as the counter acid for the organic salt formation, yielding the desired regioisomer up to 100:0. The efficient and scalable three-step protocol operates under mild conditions with a high chemo/regioselectivity, providing effective access to MMV693183.

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

BackgroundDifferentiation of induced pluripotent stem cells (iPSCs)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy. Methodsβ-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of LncRNA Malat1. Quantitative real-time PCR (qRT-PCR), Western blot (WB) analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p/Ihh were detected by dual luciferase reporter assays (LRAs). ResultsWe found that the expression of lncRNA Malat1 declined during differentiation, and overexpression (OE) of lncRNA Malat1 notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Most importantly, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments. ConclusionThis study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

Surgical Strategy for Sternal Closure in Patients with Surgical Myocardial Revascularization Using Mammary Arteries.

Coronary artery bypass grafting has evolved from all venous grafts to bilateral mammary artery (BIMA) grafting. This was possible due to the long-term patency of the left and right internal mammary demonstrated in angiography studies compared to venous grafts. However, despite higher survival rates when using bilateral mammary arteries, multiple studies report a higher rate of surgical site infections, most notably deep sternal wound infections, a so-called "never event". We designed a prospective study between 1 January 2022 and 31 December 2022 and included all patients proposed for total arterial myocardial revascularization in order to investigate the rate of surgical site infections (SSI). Chest closure in all patients was performed using a three-step protocol. The first step refers to sternal closure. If the patient's BMI is below 35 kg/m2, sternal closure is achieved using the "butterfly" technique with standard steel wires. If the patient's BMI exceeds 35 kg/m2, we use nitinol clips or hybrid wire cable ties according to the surgeon's preference for sternal closure. The main advantages of these systems are a larger implant-to-bone contact with a reduced risk of bone fracture. The second step refers to presternal fat closure with two resorbable monofilament sutures in a way that the edges of the skin perfectly align at the end. The third step is skin closure combined with negative pressure wound therapy. This system was applied to 217 patients. A total of 197 patients had bilateral mammary artery grafts. We report only 13 (5.9%) superficial SSI and only one (0.46%) deep SSI. The preoperative risk of major wound infection was 3.9 +/- 2.7. Bilateral mammary artery grafting was not associated with surgical site infection in a univariate analysis. We believe this strategy of sternal wound closure can reduce the incidence of deep surgical site infection when two mammary arteries are used in coronary artery bypass surgery.

Circulating miR-133a-3p defines a low-risk subphenotype in patients with heart failure and central sleep apnea: a decision tree machine learning approach

BackgroundPatients with heart failure with reduced ejection fraction (HFrEF) and central sleep apnea (CSA) are at a very high risk of fatal outcomes.ObjectiveTo test whether the circulating miRNome provides additional information for risk stratification on top of clinical predictors in patients with HFrEF and CSA.MethodsThe study included patients with HFrEF and CSA from the SERVE-HF trial. A three-step protocol was applied: microRNA (miRNA) screening (n = 20), technical validation (n = 60), and biological validation (n = 587). The primary outcome was either death from any cause, lifesaving cardiovascular intervention, or unplanned hospitalization for worsening of heart failure, whatever occurred first. MiRNA quantification was performed in plasma samples using miRNA sequencing and RT-qPCR.ResultsCirculating miR-133a-3p levels were inversely associated with the primary study outcome. Nonetheless, miR-133a-3p did not improve a previously established clinical prognostic model in terms of discrimination or reclassification. A customized regression tree model constructed using the Classification and Regression Tree (CART) algorithm identified eight patient subphenotypes with specific risk patterns based on clinical and molecular characteristics. MiR-133a-3p entered the regression tree defining the group at the lowest risk; patients with log(NT-proBNP) ≤ 6 pg/mL (miR-133a-3p levels above 1.5 arbitrary units). The overall predictive capacity of suffering the event was highly stable over the follow-up (from 0.735 to 0.767).ConclusionsThe combination of clinical information, circulating miRNAs, and decision tree learning allows the identification of specific risk subphenotypes in patients with HFrEF and CSA.

Maltotriose acylhydrazone surfactants: Structure-property profiles and efficient in vivo drug delivery for primary biliary cholangitis treatment

Bio-based surfactants derived from carbohydrates have attracted increasing attention due to their environmental friendliness, biodegradability, and biological functions. In this study, we synthesized a novel class of maltotriose acylhydrazone surfactants with different alkyl chain lengths (6–14 carbons) using a simple and efficient three-step protocol. We characterized their structure, surface activity, aggregation behavior, foaming properties, and emulsion stability. We found that these surfactants had high hydrophilicity, low toxicity, and good emulsifying and foaming abilities. We also investigated their potential as drug delivery vehicles for ursodeoxycholic acid (UDCA), a drug for primary biliary cholangitis (PBC). We prepared UDCA-loaded niosomes with these surfactants and evaluated their drug entrapment efficiency, in vitro drug release, and in vivo therapeutic effect in a mouse model of PBC. We demonstrated that these niosomes could efficiently encapsulate and deliver UDCA, resulting in improved liver function and reduced inflammation. Our results suggest that maltotriose acylhydrazone surfactants are promising candidates for bio-based surfactants and drug delivery systems.

Evaluation of lncRNA Expression During the Differentiation of Mesenchymal Stem Cells to Insulin-Secreting Progenitors.

Diabetes mellitus is a metabolic disease caused by a defect in insulin secretion, insulin function, or both that destroys pancreatic islet beta cells. There is ample evidence that long non-coding RNAs (lncRNAs) play a vital role in cell formation and differentiation. The present study aims to investigate the expression pattern of specific lncRNAs in mesenchymal stem cell (MSC) differentiation into insulin-producing beta cell (IPCs) progenitors for cell therapy purposes. MSCs were extracted from human umbilical cord Wharton jelly (hWJ-MSCs) using the explant method and cultured in two-dimensional (2D) and three-dimensional (3D) media on polylactic acid/Wax (PLA/Wax) nanofibrous scaffold using a three-step protocol containing CHIR99021 small molecules and Indolactam V. At the end of each differentiation step, immunocytochemistry and qRT-PCR were used to confirm the differentiation at the protein and RNA levels and the expression changes of six selective lncRNAs were evaluated by qRT-PCR. The results indicated that the expression of the selected lncRNAs was significantly altered during the differentiation process into beta progenitor cells, indicating their potential role in regulating the IPC differentiation process. More specifically, all of the desired lncRNAs demonstrated a significant increase during the beta cell differentiation, with HI-LNC71 and HI-LNA12 experiencing the highest expression in the produced Beta cell progenitors respectively (p<0.0001). These results can be valuable in tissue engineering and treatment studies by replacing beta precursor cells to control diabetic patients.

Electrospinning is a potential way for preparing edible plant protein-based nanofibers with porous surface using safflower seed meal

Food microstructure design has become an important research area in artificial food manufacturing. The fibrous structure is one of the most critical elements since it is the fundamental building blocks constructing the structure of several important food matrices. However, at present, edible fibers at nanoscale are seldomly successfully manufactured due to technical difficulties when only food-grade materials are allowed to be used. In the current study, edible nanofiber consisting of a single component of safflower seed meal protein (SMP) was prepared based on a three-step protocol consisting of electrostatic spinning, protein-cinnamaldehyde cross-linking, and subsequent immersion in the citric acid buffer to remove none-protein components. Addition of pullulan (PUL) to SMP during electrospinning facilitated fibrous structure formation. Results of SEM demonstrated that SMP: PUL = 5:5 with combined ratio 20% (w/v) in water is the upper limit to obtain well-structured composite nanofiber with no addition of Tween 80. In the second step, protein-cinnamaldehyde cross-linking was implemented to stabilize the structure of SMP. In the last step, citric acid buffer with pH4.6 was used to remove pullulan residues and thereafter nanofibers with porous surfaces composed of only SMP were successfully obtained. Our results showed that SMP single-component nanofibers had high thermal stability and good water-holding capacity, and thus suggesting that electrospinning can be a potentially innovative strategy to manufacture food fibrous microstructures especially at nanoscale.

Environmental bioavailability of arsenic, nickel and chromium in soils impacted by high geogenic and anthropogenic background contents

High arsenic, chromium and nickel in soils can pose a hazard to the ecosystem and/or human health. Large areas can be affected by elevated potentially toxic elements (PTE) background contents, entailing a significant effort for managing the potential risk. Assessing the environmental hazard associated to PTE-contaminated soils requires the determination of soil PTE environmental bioavailability, which reflects the capacity of these elements to be transferred to living organisms. Here we assess the environmental bioavailability of As, Cr and Ni in topsoils from the Liège basin and Belgian Lorraine, two areas in Wallonia, Belgium, affected by elevated As, Cr and Ni background contents. The source of soil As, Cr and Ni differs in Liège and Lorraine: anthropogenic in the former location and geogenic in the latter. The environmental bioavailability of PTE was determined using two complementary approaches: (1) by chemical fractionation with the Community Bureau of Reference (BCR) three-step sequential extraction protocol and (2) by estimating the phytoavailability using a plant-based biotest (Lolium multiflorum as plant model). The results show that total As (6–130 mg·kg−1), Cr (15–268 mg·kg−1), and Ni (8–140 mg·kg−1) contents in the Liège and Lorraine soils frequently exceed the soil clean-up standards. However, no positive correlation was found between the total contents and BCR extraction results or rye-grass contents, except for As in Liège soils. Total As, Cr or Ni contents surpassing soil standards do not necessarily result in elevated mobile, potentially mobilizable and phytoavailable contents. In general, environmental bioavailability of As, Cr and Ni is higher in soils from Liège basin compared to those sampled in Belgian Lorraine. The mobile and potentially mobilizable fractions of As, Cr and Ni account for <30 % of their total contents following the BCR extractions. Our study provides valuable information for sustainable management at the regional scale of soils containing high PTE contents.

Deep neural network analysis models for complex random telegraph signals

Time-fluctuating signals are ubiquitous and diverse in many physical, chemical, and biological systems, among which random telegraph signals (RTSs) refer to a series of instantaneous switching events between two discrete levels from single-particle movements. A reliable RTS analysis is a crucial prerequisite to identify underlying mechanisms related to device performance and sensitivity. When numerous levels are involved, complex patterns of multilevel RTSs occur and make their quantitative analysis exponentially difficult, hereby systematic approaches are often elusive. In this work, we present a three-step analysis protocol via progressive knowledge-transfer, where the outputs of the early step are passed onto a subsequent step. Especially, to quantify complex RTSs, we resort to three deep neural network architectures whose trained models can process raw temporal data directly. We furthermore demonstrate the model accuracy extensively with a large dataset of different RTS types in terms of additional background noise types and amplitude size. Our protocol offers structured schemes to extract the parameter values of complex RTSs as imperative information with which researchers can draw meaningful and relevant interpretations and inferences of given devices and systems.

Purification and Molecular Characterization of Fucoidan Isolated from Ascophyllum nodosum Brown Seaweed Grown in Ireland.

The present study investigates the molecular characteristics of fucoidan obtained from the brown Irish seaweed Ascophyllum nodosum, employing hydrothermal-assisted extraction (HAE) followed by a three-step purification protocol. The dried seaweed biomass contained 100.9 mg/g of fucoidan, whereas optimised HAE conditions (solvent, 0.1N HCl; time, 62 min; temperature, 120 °C; and solid to liquid ratio, 1:30 (w/v)) yielded 417.6 mg/g of fucoidan in the crude extract. A three-step purification of the crude extract, involving solvents (ethanol, water, and calcium chloride), molecular weight cut-off filter (MWCO; 10 kDa), and solid-phase extraction (SPE), resulted in 517.1 mg/g, 562.3 mg/g, and 633.2 mg/g of fucoidan (p < 0.05), respectively. In vitro antioxidant activity, as determined by 1,1-diphenyl-2-picryl-hydrazyl radical scavenging and ferric reducing antioxidant power assays, revealed that the crude extract exhibited the highest antioxidant activity compared to the purified fractions, commercial fucoidan, and ascorbic acid standard (p < 0.05). The molecular attributes of biologically active fucoidan-rich MWCO fraction was characterised by quadruple time of flight mass spectrometry and Fourier-transform infrared (FTIR) spectroscopy. The electrospray ionisation mass spectra of purified fucoidan revealed quadruply ([M+4H]4+) and triply ([M+3H]3+) charged fucoidan moieties at m/z 1376 and m/z 1824, respectively, and confirmed the molecular mass 5444 Da (~5.4 kDa) from multiply charged species. The FTIR analysis of both purified fucoidan and commercial fucoidan standard exhibited O-H, C-H, and S=O stretching which are represented by bands at 3400 cm-1, 2920 cm-1, and 1220-1230 cm-1, respectively. In conclusion, the fucoidan recovered from HAE followed by a three-step purification process was highly purified; however, purification reduced the antioxidant activity compared to the crude extract.

A Three-Step Taxane Titration Protocol Decreases Hypersensitivity Reactions During First and Second Exposures.

Patients receiving taxanes are at risk for developing hypersensitivity reactions (HSRs) primarily during first and second lifetime exposures. Immediate HSRs require emergency care and can interfere with the continuation of preferred treatment. Although different approaches to slow titration have been used successfully for desensitization after HSR occurrence, there are no standardized recommendations for taxane titration to prevent HSRs. To determine if a gradual, three-step infusion rate titration decreases the rate and severity of immediate HSRs during first and second lifetime exposures to paclitaxel and docetaxel. We used a prospective, interventional design with historical comparisons to evaluate a sample of 222 first and second lifetime exposure paclitaxel and docetaxel infusions. The intervention was a three-step infusion rate titration provided at the initiation of first and second lifetime exposures. Ninety-nine titrated infusions were compared with 123 historical records of nontitrated infusions. Compared with the nontitrated group (n = 123), the titrated group (n = 99) had significantly less HSRs (19% v 7%; P = .017). No significant difference in HSR severity was found between groups (P = 1.00). However, four nontitrated patients received epinephrine, and one required transfer to the emergency department (ED) because of reaction severity. In contrast, no titrated patients received epinephrine or required transfer to the ED. In the nontitrated group, seven patients did not complete their infusions versus one patient in the titrated group. A standardized, three-step infusion rate titration prevented HSR occurrence. Significant issues affecting practice feasibility and sustainability were addressed.

Effect of a Change in the CaCl2/Pectin Mass Ratio on the Particle Size, Rheology and Physical Stability of Lemon Essential Oil/W Emulgels

A three-step (rotor-stator-microfluidization-rotor stator) protocol was used to prepare 15% lemon essential oil in water emulgels using a mixture of Tween 80 and Span 20 surfactants as low molecular mass emulsifiers and 0.4% low-methoxyl citrus peel pectin as a gelling agent. Ca2+ was used as a gel-promoting agent. Different CaCl2/pectin mass ratio values from 0.3 to 0.7 were used. Emulgels showed a microstructure consisting of oil droplets embedded in a sheared gel matrix, as demonstrated by bright field optical microscopy. Laser diffraction tests showed multimodal particle size distributions due to the coexistence of oil droplets and gel-like particles. Multiple light scattering tests revealed that the physical stability of emulgels was longer as the CaCl2/pectin mass ratio decreased and that different destabilization mechanisms took place. Thus, incipient syneresis became more important with increasing CaCl2 concentration, but a parallel creaming mechanism was detected for CaCl2/pectin mass ratio values above 0.5. Dynamic viscoelastic and steady shear flow properties of the emulgels with the lowest and highest CaCl2/pectin mass ratio values were compared as a function of aging time. The lowest ratio yielded an emulgel with enhanced connectivity among fluid units as indicated by its wider linear viscoelastic region, higher storage modulus, loss modulus and viscosity values, and more shear thinning properties than those of the emulgel formulated with the highest CaCl2/pectin mass ratio. The evolution of the dynamic viscoelastic properties with aging time was consistent with the information provided by monitoring scans of backscattering as a function of sample height.

A three step protocol for the development of an innovative footwear (shoe and sensor based insole) to prevent diabetic foot ulceration.

The incidence of diabetic foot ulceration (DFU) is increasing worldwide. Therapeutic footwear is usually recommended in clinical practice for preventing foot ulcers in persons with diabetes. The project Science DiabetICC Footwear aims to develop innovative footwear to prevent DFU, specifically a shoe and sensor-based insole, which will allow for monitoring pressure, temperature, and humidity parameters. This study presents a three-step protocol for the development and evaluation of this therapeutic footwear, specifically: (i) a first observational study will specify the user requirements and contexts of use; (ii) after the design solutions were developed for shoe and insole, the semi-functional prototypes will be evaluated against the initial requirements; (iii) and a pre-clinical study protocol will enable the evaluation of the final functional prototype. The eligible diabetic participants will be involved in each stage of product development. The data will be collected using interviews, clinical evaluation of the foot, 3D foot parameters and plantar pressure evaluation. This three-step protocol was defined according to the national and international legal requirements, ISO norms for medical devices development, and was also reviewed and approved by the Ethics Committee of the Health Sciences Research Unit: Nursing (UICISA: E) of the Nursing School of Coimbra (ESEnfC). The involvement of end-users (diabetic patients) will enable the definition of user requirements and contexts of use to develop design solutions for the footwear. Those design solutions will be prototyped and evaluated by end-users to achieve the final design for therapeutic footwear. The final functional prototype will be evaluated in pre-clinical studies to ensure that the footwear meets all the requirements to move forward to clinical studies. The three-step study outlined in this protocol will provide the necessary insights during the product development, ensuring this new therapeutic footwear's main functional and ergonomic features for DFU prevention.

2-{[4-(4-Bromophenyl)piperazin-1-yl)]methyl}-4-(3-chlorophenyl)-5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione

The novel compound 2-{[4-(4-bromophenyl)piperazin-1-yl)]methyl}-4-(3-chlorophenyl-5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione is obtained in good yield via a three-step protocol. The product’s structure is assigned by HRMS, IR, 1H and 13C NMR experiments.

Efficient extraction of small microplastic particles from rat feed and feces for quantification

To date, microplastic is ubiquitously encountered in the environment. Studies analyzing microplastic in terrestrial ecosystems, including animal feces and feed, are few. Microplastic quantification method validation and harmonization are not yet far developed. For the analysis of small microplastic, approximately <0.5 mm, extraction from organic and inorganic materials is fundamental prior to quantitative and qualitative analysis. Method validation, including recovery studies, are necessary throughout the analytical chain. In this study, we developed an optimized, efficient protocol with a duration of 72 h for the digestion of laboratory rat feed and feces. A combination of a mild acidic (H2O2 15%; HNO3 5%) and an alkaline treatment (10% KOH) dissolving the previous filter, followed by enzymatic digestion (Viscozyme®L) proved to be efficient for the extraction and identification of spiked polyamide (15–20 μm) and polyethylene (40–48 μm) from feed and feces samples from rats, showing high recovery rates. Extracted rat feces samples from an in vivo study in which Wistar rats were fed with feed containing microplastic were analyzed with pyrolysis-gas chromatography-Orbitrap™ mass spectrometry, quantifying recovered microplastic in rat feces in environmentally relevant concentrations. The presented three-step protocol provides a suitable, time and cost-effective method to extract microplastic from rat feed and feces.

Fractionation of Native Protein Complexes from Mammalian Cells to Determine the Differential Proteasome Activity and Abundance.

Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines. Key features • Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes. • Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix. • In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes' abundance using antibodies.

Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus merguiensis).

Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments. We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86-100%) and 100% specificity (95% CI: 66-100%) for detection of HPV. The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

Insulin-producing cell clusters derived from human gingival mesenchymal stem cells as a model for diabetes research.

The human gingiva-derived mesenchymal stem cells (hGMSCs) possess a great potential to develop the cell-based therapy for diabetes due to its unscarred healing capacity and reparative potential. In this current study, we isolated, cultured and characterised the GMSCs and explored their potential to differentiate into Insulin Producing Cell Clusters (IPCCs). The cells derived from gingival tissues exhibited fibroblast-like morphology. The flow cytometric analysis revealed positive expression of CD73(97.43%), CD90(95.05%), and CD105(93.17%) and negative expression of CD34(0.05%), CD45(0.09%), and HLA-DR (0.025) surface markers. We then converted this adherent fibroblast-like GMSCs into floating IPCCs using a sequential three-step protocol containing a different combination of differentiating agents. Initially, the presence of insulin in IPCCs was confirmed by dithizone staining. Glucose-stimulated insulin secretion (GSIS) assay confirmed that IPCCs secrete insulin in response to glucose. Generated IPCCs express pancreatic markers such as insulin, pdx1, glucagon, GLUT4 and GLUT2 as evidenced by RT-PCR analysis. Our results unequivocally showed that IPCCs can be generated from gingiva which is a potential source of postnatal MSCs. Our results offer the IPCCs generated from hGMSCs a platform for screening anti-diabetic drugs and a new autologous source of tissue for islet transplantation for the treatment of diabetes. Our results unequivocally demonstrate for the first time that hGMSCs can be used as an attractive non-invasive tissue source for generating IPCCs, which can be employed in diabetes research for screening antidiabetic agents and also for transplantation in type 1 diabetic patients as autologous source without the need of immunosuppression.

Dihydropyrazinoquinazolinones via SN2 Sulfamidate Ring-Opening and a Sequential Quinazolinone-Amidine Rearrangement Strategy (SQuAReS).

A synthesis of dihydropyrazino-[2,1-b]-quinazolinones is described using a 2-alkylaminoquinazolinone-mediated ring opening of a-/chiral sulfamidates, followed by a tandem quinazolinone-amidine rearrangement termed SQuAReS. This approach takes advantage of sulfamidates whose regioselective ring opening, after hydrolysis, appends an optimally distanced nucleophilic amine to a quinazolinone such that subsequent domino rearrangements are favored, integrating unique substitution patterns on a privileged core. This three-step protocol integrated five telescoped transformations and generated 20 pyrazinoquinazolinones in up to 74% yield with high enantiomeric fidelity and diastereoselectivity.

Single step "See and Treat" strategy might be replacing the "conventional three step strategy" in management of preinvasive cervical lesions at tertiary center: A North Indian study.

The aim of this study was to compare overtreatment rates of see and treat colposcopy-based single step protocol with cytology and colposcopy-guided biopsy-based conventional three-step protocol using loop electrosurgical excision procedure (LEEP) for treatment of preinvasive lesions of cervix. Prospective interventional study was carried out over a period of 1 year. Recruitment of cases was done from the 664 diagnostic colposcopies performed for various gynecological indications. Among 496 colposcopies performed exclusively for unhealthy cervix on per speculum examination, 74 women had high-grade colposcopy (Swede score ≥5). Subsequently, 50 women were enrolled under the see and treat arm, arm 1 and underwent LEEP. In study arm 2, conventional three-step strategy, concurrently 22 women with abnormal cytology. ≥ Atypical squamous cells of undetermined significance and unhealthy cervix were enrolled for colposcopy and if indicated, guided biopsy was obtained and tissue was sent for histopathology. Only 12 such women having HPE reports of cervical intraepithelial neoplasia (CIN) 2 or 3 were subjected to LEEP. Overtreatment was defined as CIN 1 or less on final LEEP tissue histopathology. The overtreatment rate in See and Treat protocol was 44% when colposcopy Swede score cutoff was considered 5, which fell down to 0% when Swede score cutoff was taken 7. Conventional three step protocol had an overtreatment rate of 8.3%. Incidentally diagnosed high-grade CIN or invasive cancer was found in 24%. Discrepancy between biopsy tissue and LEEP tissue histopathology was 50% in conventional arm. Women with unhealthy cervix having high-grade colposcopy (Swede score ≥7) can be directly subjected to LEEP without waiting for results of any initial screening modality. Advantages include minimal over treatment coupled with reduced patient visits and interventions.