Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis.

Chlamydia trachomatis ( C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/µL to 1×10 -3pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae.

Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/µL to 1 × 10-3 pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/µL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/µL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/µL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/µL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Development and Clinical Validation of Iso-IMRS: A Novel Diagnostic Assay for P. falciparum Malaria.

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/µL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.

Pharmaceutical Co-Crystallization: Regulatory Aspects, Design, Characterization, and Applications.

Pharmaceutical co-crystals are novel class of pharmaceutical substances, which possess an apparent probability of advancement of polished physical properties offering stable and patentable solid forms. These multi-component crystalline forms influence pertinent physicochemical parameters like solubility, dissolution rate, chemical stability, physical stability, etc. which in turn result in the materials with superior properties to those of the free drug. Co-crystallization is a process by which the molecular interactions can be altered to optimize the drug properties. Co-crystals comprise a multicomponent system of active pharmaceutical ingredient (API) with a stoichiometric amount of a pharmaceutically acceptable coformer incorporated in the crystal lattice. By manufacturing pharmaceutical co-crystals, the physicochemical properties of a drug can be improved thus multicomponent crystalline materials have received renewed interest in the current scenario due to the easy administration in the pharmaceutical industry. There is an immense amount of literature available on co-crystals. However, there is a lack of an exhaustive review on a selection of coformers and regulations on co-crystals. The review has made an attempt to bridge this gap. The review also describes the methods used to prepare co-crystals with their characterization. Brief description on the pharmaceutical applications of co-crystals has also been incorporated here. Efforts are made to include reported works on co-crystals, which further help to understand the concept of co-crystals in depth.

Genome Mining-Based Identification of Identical Multirepeat Sequences in Plasmodium falciparum Genome for Highly Sensitive Real-Time Quantitative PCR Assay and Its Application in Malaria Diagnosis.

Developing ultrasensitive methods capable of detecting submicroscopic parasitemia-a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence-is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and explore their potential as primers for PCR. Real-time quantitative PCR analyses have shown the ability of P. falciparum IMRSs to amplify as low as 2.54 fg of P. falciparum genomic DNA (approximately 0.1 parasite), with a striking 100-fold increase in detection limit when compared with P. falciparum 18S rRNA (251.4 fg; approximately 10 parasites). Validation with clinical samples from malaria-endemic regions has shown 6.70 ± 1.66 cycle better detection threshold in terms of Ct value for P. falciparum IMRSs, with approximately 100% sensitivity and specificity. Plasmodium falciparum IMRS assays are also capable of detecting submicroscopic infections in asymptomatic samples. To summarize, this approach of initiating amplification at multiple loci across the genome and generating more products with increased analytical sensitivity is different from classic approaches amplifying multicopy genes or tandem repeats. This can serve as a platform technology to develop advanced diagnostics for various pathogens.

Effectiveness of Electrostimulation on Whole Salivary Flow Among Patients with Type 2 Diabetes Mellitus.

CONTEXT: Xerostomia and hyposalivation are associated with diabetes. Research is sparse regarding electrostimulation as a mainstream therapy for salivary gland hypofunction. OBJECTIVE: To clinically evaluate the effectiveness of transcutaneous electric nerve stimulation (TENS) therapy in stimulating whole salivary flow among patients with xerostomia and hyposalivation caused by diabetes mellitus. DESIGN: Forty patients between age 30 to 75 years with diabetes mellitus categorized as controlled or uncontrolled who had subjective symptoms of xerostomia and an objective sign of hyposalivation were included in a prospective study. MAIN OUTCOME MEASURES: Unstimulated saliva through the "low forced spitting" method and stimulated saliva collection using TENS were assessed and compared. Longer-term effects of TENS application were evaluated by recalling the patient 24 hours later. RESULTS: A statistically significant increase in stimulated whole saliva after TENS application in continuous mode (p < 0.001) was demonstrated compared with unstimulated saliva, especially in xerostomic patients with diabetes. Burst mode inferred a statistically significant decrease in salivary flow (p < 0.001). CONCLUSION: In patients with diabetes with xerostomia and hyposalivation, TENS was highly effective in stimulating whole salivary flow.

Intramyometrial Injection of Vasopressin: A Novel Method for Hemostasis at Laparoscopic Management of Cornual Ectopic.

STUDY OBJECTIVE: Although all ectopic pregnancies are associated with risk of hemorrhage, cornual pregnancies are feared for catastrophic hemorrhage and uncontrollable bleeding. The maternal mortality rate can be as high as 2.5%, which is 7 times higher than the mortality rate for ectopic pregnancies in general. Different techniques have been used to control hemostasis, including purse string suture, square suture, endo-loop, electrocoagulation, and devascularization. Injection of vasopressin into the uterus is a simple method that greatly reduces the blood loss at cornuostomy. DESIGN: A step by step demonstration of the surgical procedure (Canadian Task Force classification III). SETTING: Hospital. PATIENT, INTERVENTIONS, MEASUREMENTS AND MAIN RESULTS: A 32-year-old woman, G7P3L3A3 (Gravida 7, Para 3, Living 3, Abortion 3), with 4 months of amenorrhea, was diagnosed with a cornual ectopic pregnancy. She was treated with 200 mg mifepristone and 800 µg misoprostol. She had undergone dilatation and curettage twice and was referred for persisting cornual ectopic pregnancy. At laparoscopy, 20 U injection vasopressin in 100 mL .9% normal saline was injected into the myometrium of the uterus. Incision was made over the ectopic pregnancy with ultrasonic energy and the ectopic pregnancy enucleated using suction apparatus and ultrasonic energy. Hemostasis was ensured and the bed sutured with barbed suture. Surgery duration was 1 hour, and blood loss was 200 mL. Institutional review board and ethics committee approval was obtained. CONCLUSION: Injection of vasopressin into the uterus significantly reduces blood loss, operative time, and patient morbidity and mortality at laparoscopic cornuostomy.

Functional skeletal muscle regeneration from differentiating embryonic stem cells.

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-alpha receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

Surface chemistry influences implant-mediated host tissue responses.

Implant-mediated fibrotic reactions are detrimental to the performance of encapsulated cells, implanted drug release devices, and sensors. To improve the implant function and longevity, recent research has emphasized the need for inducing alterations in cellular responses. Although material surface functional groups have been shown to be potent in affecting cellular activity in vitro and short-term in vivo responses, these groups appear to have little influence on long-term in vivo fibrotic reactions, possibly as a result of insufficient interactions between recruited host cells and functional groups on the implants. To maximize the influence of functionality on cells, and to mimic drug release microspheres, functionalized micron-sized particles were created and tested for their ability in modulating tissue responses to biomaterial implants. In this work, the surfaces of polypropylene particles were controllably coated with four different functional groups, specifically -OH, -NH(2), -CF(x), and -COOH, using a radio frequency glow discharge plasma polymerization technique. The effect of these surface functionalities on host tissue responses were then evaluated using a mice subcutaneous implantation model. Major differences were observed in contrasting tissue response to the different chemistries. Surfaces with -OH and -NH(2) surface groups induced the thickest fibrous capsule accompanied with the greatest cellular infiltration into the implants. In contrast, surfaces with -CF(x) and -COOH exhibited the least inflammatory/fibrotic responses and cellular infiltrations. The present results clearly demonstrate that, by increasing the available functionalized surface area and spatial distribution, the effect of surface chemistry on tissue reactivity can be substantially enhanced.