Descemeta membrānas endotēlija keratoplastika: nākamās paaudzes sekvenēšanas metagenomiski optimizētu audu sagatavošana un ķirurģijas tehnika. Promocijas darba kopsavilkums

Pētījumā tika analizēta optimizācija, pielietojot metagenomiku radzenes audu saglabāšanā. Nosaukums: Nākamās paaudzes sekvencēšana mikroorganismu noteikšanai cilvēka donora radzenes audu saglabāšanas vidē. Mērķis: Ar nākamās paaudzes sekvencēšanas metodi noteikt mikroorganismu klātbūtni cilvēka donora radzenes audu uzglabāšanas vidē. Materiāli un metodes: Kopā tika izmantoti septiņi paraugi no orgānu kultūras (no angļu val. – organ culture (OC)) grupas (Cornea Max, Eurobio, Les Ulis, Francija) ar vienu kontroli (sterila barotne bez radzenes audiem) un septiņi paraugi no hipotermiskās uzglabāšanas grupas (Cornea Cold, Eurobio) ar vienu kontroli. Pirms paraugu savākšanas radzenes audus ievietoja attiecīgajās uzglabāšanas vietās uz 14 dienām. Uzglabāšanas vide (2 ml) no katra parauga tika ievietota mēģenēs, kas nesatur RNS-āzi, un tika nosūtīta 16S un 18S ribosomu RNS sekvencēšanai. Vienlaikus 1 ml barotnes parauga tika izmantots konvencionālajai diagnostikas metodei (no angļu val. – conventional diagnostic method (CDM)), izmantojot Bactec instrumentus. Rezultāti: Gan OC, gan hipotermiskās uzglabāšanas un kontroles paraugos visbiežāk novēroja Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas un Nitrobacter ģintis. Acidovorax, Acetobacter un Hydrogenophilus ģintis tika atklātas galvenokārt hipotermiskās uzglabāšanas grupā. Visbiežāk konstatētais sēņu patogēns piederēja Malassezia ģintij, kas tika atrasts abās uzglabāšanas vidēs. Ar CDM visi paraugi bija negatīvi attiecībā uz mikroorganismiem. Secinājumi: Metagenomika nodrošina pilnīgu taksonomisko profilu noteikto organismu genomiskajam materiālam, tādējādi sniedzot daudz plašāku mikrobioloģiskās diagnostikas pieeju nekā CDM. Metodes izmaksu un apgrozījuma laika dzīvotspējīgu organismu noteikšanai samazināšana palīdzētu šo tehnoloģiju ieviest ikdienas klīniskajā praksē.

The Accumulation of Essential Oils In Organ Culture of Acinos Graveolens Via Methyl Jasmonate And Salicylic Acid Exertion

The impact of Salicylic acid (SA) and Methyl jasmonate (MJ) on growth, and essential oil quantity and quality in organ culture of Acinos graveolens were investigated. In the present study, nodal segments were used as explants for shoot production using MS media with BA and NAA. Afterward, they were transferred to liquid MS media culture containing SA and MJ with 50, 100, and 150 µM and their combinations (SA25+MJ25, SA50+MJ25, SA25+MJ50, and SA50+MJ50). After three weeks, samples were collected to assess the morphological and some growth parameters, quantitative changes in pigment amounts as well as volatile contents. Accordingly, almost all treatments led to a notable decrease in growth parameters in comparison with control. The highest amount of Chl a, b and carotenoids were achieved by MJ100, SA50, and SA25+MJ50, respectively. In addition, GC-MS results indicated 38 components of volatile products mainly alkane hydrocarbons and sesquiterpenes. The major identified compounds were Decane, Eicosane, and Germacrene D. Altogether, results asserted that SA25+MJ25 and SA100 were more efficient in the enhancement of essential oil content among all treatments.

Ras signaling and its effector RREB1 are required for the dissociation of MEE cells in palatogenesis

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has largely remained unelucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for the proper MEE removal. Ras Responsive Element Binding Protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental EMT, in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Functional Significance of Selective Expression of ERα and ERβ in Mammary Gland Organ Culture

Thoracic pair of mammary glands from steroid hormone-pretreated mice respond to hormones structurally and functionally in organ culture. A short exposure of glands for 24 h to 7,12 Dimethylbenz(a)anthracene (DMBA) during a 24-day culture period induced alveolar or ductal lesions. Methods: To differentiate the functional significance of ERα and ERβ, we employed estrogen receptor (ER) knockout mice. We compared the effects of DMBA on the development of preneoplastic lesions in the glands in the absence of ERα (αERKO) and ERβ (βERKO) using an MMOC protocol. Glands were also subjected to microarray analyses. We showed that estradiol can be replaced by EGF for pretreatment of mice. The carcinogen-induced lesions developed under both steroids and EGF pretreatment protocols. The glands from αERKO did not develop any lesions, whereas in βERKO mice in which ERα is intact, mammary alveolar lesions developed. Comparison of microarrays of control, αERKO and βERKO mice showed that ERα was largely responsible for proliferation and the MAP kinase pathways, whereas ERβ regulated steroid metabolism-related genes. The results indicate that ERα is essential for the development of precancerous lesions. Both subtypes, ERα and Erβ, differentially regulated gene expression in mammary glands in organ cultures.

Gram stain and addition of amphotericin B to improve the microbial safety of human donor corneas

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015–2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015–2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1–3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections.