Cerebral organoids: a promising model in cellular technologies

The development of the human brain is a complex multi-stage process including the formation of various types of neural cells and their interactions. Many fundamental mechanisms of neurogenesis have been established due to the studying of model animals. However, significant differences in the brain structure compared to other animals do not allow considering all aspects of the human brain formation, which could play the main role in the development of unique cognitive abilities for human. Four years ago, Lancaster’s group elaborated human pluripotent stem cell-derived three-dimensional cerebral organoid technology, which opened a unique opportunity for researchers to model early stages of human neurogenesis in vitro. Cerebral organoids closely remodel many endogenous brain regions with specific cell composition like ventricular zone with radial glia, choroid plexus, and cortical plate with upper and deeper-layer neurons. Moreover, human brain development includes interactions between different brain regions. Generation of hybrid three-dimensional cerebral organoids with different brain region identity allows remodeling some of them, including long-distance neuronal migration or formation of major axonal tracts. In this review, we consider the technology of obtaining human pluripotent stem cell-derived three-dimensional cerebral organoids with different modifications and with different brain region identity. In addition, we discuss successful implementation of this technology in fundamental and applied research like modeling of different neurodevelopmental disorders and drug screening. Finally, we regard existing problems and prospects for development of human pluripotent stem cell-derived threedimensional cerebral organoid technology

Evalution of endogenous intoxication in patients on herpesassociated multiforme exudative erythema

In this work the determination of the parameters of endogenous intoxication in patients herpes associated multiforme exudative erythema in the context of the severity of the disease compared with patients with exudative erythema multiforme other etiologies and patients with recurrent herpes simplex. Shown that patients herpes associated multiform exudative erythema of the oral cavity have a pronounced degree of endogenous intoxication, which is directly correlated with the degree of severity of the disease, which gives grounds for inclusion in the scheme of complex treatment of patients with herpeszoster bahatoformatne exudative erythema methods of detoxication therapy

CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression

Germline-restricted chromosome (GRC) is widespread among songbirds

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages

Напитки и сладкие блюда, рекомендуемые больным дистрофической миотонией с орофарингеальной дисфагией

Myotonic dystrophy is a multisystemic disease which mutation may influence your development and function of different organs and tissue: smooth and skeletal-muscular tissue; heart; organs of the eye, brain. This review gives a simple information about balanced diet of patients myotonic dystrophy with oropharyngeal dysphagia.Дистрофическая миотония (ДM; англ. сongenital myotonic dystrophy, myotonic dystrophy) является наследственным мультисистемным заболеванием, при котором мутация затрагивает развитие и функционирование различных органов и тканей: гладкой и скелетной мышечной ткани, сердца, органа зрения (глаза), головного мозга. В представленном обзоре обобщены доступные сведения по вопросам рационального питания больных дистрофической миотонией с орофарингеальной дисфагией

Cytotoxic effect of the VVGMCSF-Lact oncolytic virus against 3D cultures of human glioblastoma cells U-87 MG

Background. One of the promising methods of treating tumors is virotherapy, which is based on direct lysis of cancer cells by a virus and a virus-mediated antitumor immune response of the body. For the recombinant vaccinia virus strain VVGMCSF-Lact, producing human GMCSF and the oncotoxic protein lactaptin, cytotoxic and antitumor effects were shown in experiments in vitro and in vivo, respectively, when using adhesive cultures of U-87 MG human glioblastoma cells. 3D cultures are a more relevant tumor model than adhesive models, as they more fully reflect the realistic scenario of cancer development, as well as the response of the tumor to anticancer therapy.The aim. To evaluate the cytotoxic effect of the oncolytic virus VV-GMCSF-Lact against 3D cultures of human glioblastoma U-87 MG.Materials and methods. The following methods were used in the work: cultivation of 3D cell cultures, cytofluorometry, microscopic analysis, virus titration, statistical analysis.Results. U-87 MG cells were transduced with a lentiviral vector carrying the GFP reporter gene. The cytotoxicity of the VV-GMCSF-Lact virus (IC50) against the studied cells was 0.024 PFU/cell. U-87 MG cells were cultured under conditions for the formation of 3D structures. Microscopic analysis showed the oncolytic effect of the virus on the cells of 3D cultures as early as 24 hours after the start of incubation. Flow cytometry showed an increase in the granularity of glioblastoma cells under the action of the virus, which indicates active replication of the virus in the cells. The virus titer was 0.44 PFU/cell.Conclusions. The recombinant VV-GMCSF-Lact virus has a cytotoxic effect on 3D human glioblastoma U-87 MG cell cultures and actively replicates in them. In the future, to test the oncolytic effect of VV-GMCSF-Lact, it is planned to use not only 3D human glioblastoma cultures, but also cerebral organelles obtained in the process of cocultivation of glioblastoma cells and induced human pluripotent cells

Генетична оцінка схильності до гіпертрофічної форми гінгівіту у дітей

The study is dedicated to searching for genetic factors that determine the predisposition to children's hypertrophic and catarrhal forms of gingivitis. The study involved children with hypertrophic gingivitis (12 patients) and chronic catarrhal gingivitis (16 patients). Because of the study, it was found that hypertrophic gingivitis and chronic catarrhal gingivitis differed significantly in the distribution of polymorphism genotypes TNFRSF1B, MMP1B and TGFB1 in examined patients. No difference was found between the groups by rs1800629 TNF-alpha-308G>A, rs1800795 IL6-174G>C, rs3024491 IL10-1082G>A and rs1800012 COL1A1 IVS1 2046G>T polymorphisms. The results obtained, in our opinion, should be taken into account in the development of treatment and prevention measures to support the dental treatment of children with hypertrophic gingivitis.Дослідження присвячено пошуку генетичних чинників, що визначають схильність до гіпертрофічної та катаральної форм гінгівіту у дітей. В дослідженні приймали участь групи дітей з гіпертрофічним гінгівітом (12 пацієнтів) та з хронічним катаральним гінгівітом (16 пацієнтів). У результатах проведеного дослідження було виявлено, що гіпертрофічний гінгівіт і хронічний катаральний гінгівіт суттєво відрізнялися за розподілом генотипів поліморфізму TNFRSF1B, MMP1B і TGFB1 у обстежених пацієнтів. Не було знайдено будь-якої різниці між групами за поліморфізмами rs1800629 TNF-alpha-308G>A, rs1800795 IL6-174G>C, rs3024491 IL10-1082G>A та rs1800012 COL1A1 IVS1 2046G>T. Отримані результати, на наш погляд, необхідно враховувати при розробці лікувально-профілактичних заходів супроводження стоматологічного лікування дітей з гіпертрофічним гінгівітом

Изменения иммунного статуса больных герпесассоцированной многоформной экссудативной эритремой после проведенного лечения

У статі наведено зміни імунного статусу хворих на герпесасоційовану багатоформну ексудативну еритему (ГА БЕЕ) під впливом патогенетично обгрунтованого методу комплексного лікування із включенням препарату Лікопід місцево, Ентеросгель (місцево та ентерально) і Ацикловір. Показано, що включення до комплексного лікування рецидивів ГА БЕЕ імуномодулюючого препарату Лікопід на фоні системної детоксикаційної терапії Ентеросгелем дає можливість більш ефективно досягти позитивної динаміки в регуляції клітинної та гуморальної ланок імунітету.The article presents the changes of the immune status of patients to herpes-associated multiform exudative erythema (HA MEE) under the influence of pathogenetically grounded method of complex treatment with the inclusion of the drug Likopid locally, Enterosgel (topically and enteral) and Acyclovir. It is shown that the inclusion in the complex treatment of relapses of GA BEE immunomodulating drug Lycopid on the background of systemic detoxification therapy with Enterosgel makes it possible to more effectively achieve positive dynamics in the regulation of cellular and humoral immunity.В статье приведены изменения иммунного статуса больных герпесассоцированной многоформной экссудативной эритемой (ГА МЭЭ) под влиянием патогенетически обоснованного метода комплексного лечения с включением препарата Ликопид местно, Энтеросгель (местно и энтерально) и Ацикловир. Показано, что включение в комплексное лечение рецидивов ГА МЭЭ иммуномодулирующего препарата Ликопид на фоне системной детоксикационной терапии Ентеросгелем дает возможность более эффективно достичь положительной динамики в регуляции клеточного и гуморального звеньев иммунитета

Вплив кормової добавки “Метісевіт плюс” на активність глутатіонової системи організму бугайців за умов техногенного навантаження

Pollution of the environment with heavy metals due to artificial activities of the population has led to several problems for agricultural production. The work aims to study the influence of Metisevit Plus feed additive on the state of the glutathione system of the body of bulls under conditions of man-caused load. The research was conducted based on the private agricultural enterprise “Ukraine” Dubrovytsia district of Rivne region on 12 bulls six months old, Ukrainian black-and-white dairy breed, which was formed into two groups of 6 animals each: 1 group – control, bulls were on a standard diet; Group 2 – experimental, bulls were fed a feed additive “Metisevit plus” at a dose of 0.5 g/kg of feed. According to studies, cadmium and lead load reduce the activity of the glutathione link in the antioxidant defense system of bulls. Feeding the Metisevit Plus feed additive to the bulls of the experimental group for 30 days helped increase the level of non-enzymatic and enzymatic activity of the glutathione system. A probable increase in the level of reduced glutathione was found on 30 and 40 days of the experiment, were compared with the control group of animals, it increased by 7.2 and 7.1 %, respectively. In the study of the activity of glutathione peroxidase and glutathione reductase, it was found that in these study periods they ranged from 35.7 ± 1.61 and 35.3 ± 1.55 nmol NADPH/min per 1 mg of protein (P ≤ 0.01) and 1.67 ± 0.041 and 1.64 ± 0.039 nmol NADPH/min per 1 mg of protein (P ≤ 0.001). Thus, studies on bulls indicate that the feed additive Metisevit Plus, when fed to bulls, under a load of cadmium and lead, contributed to the activation of the glutathione antioxidant defense system by increasing the level of enzymatic and non-enzymatic units. Our studies confirm the feasibility of using a feed additive, Metisevit Plus, to prevent lead-cadmium toxicosis.Забруднення довкілля важкими металами внаслідок техногенної діяльності населення призвело до низки проблем для сільськогосподарського виробництва. Мета роботи – вивчити вплив кормової добавки “Метісевіт плюс” на стан глутатіонової системи організму бугайців за умов техногенного навантаження. Дослідження проводились на базі сільськогосподарського приватного підприємства “Україна” Дубровицького району Рівненської області на 12 бугайцях шестимісячного віку, української чорно-рябої молочної породи, які були сформовані у 2 групи по 6 тварин у кожній: 1 група – контрольна (К), бугайці перебували на стандартному раціоні; 2 група – дослідна (Д), бугайцям згодовували кормову добавку “Метісевіт плюс” у дозі 0,5 г/кг комбікорму. Згідно з проведеними дослідженнями встановлено, що за кадмієвого і свинцевого навантаження знижується активність глутатіонової ланки системи антиоксидантного захисту оргнізму бугайців. Згодовування кормової добавки “Метісевіт плюс” бугайцям дослідної групи протягом 30 діб сприяло підвищенню рівня неензимної та ензимної активності глутатіонової системи. Вірогідне підвищення рівня відновленого глутатіону виявляли на 30 і 40 доби досліду, де порівняно з контрольною групою тварин він зріс на 7,2 і 7,1 % відповідно. При дослідженні активності глутатіонпероксидази та глутатіонредуктази встановлено, що у вказані періоди досліджень вони коливалися у межах величин 35,7 ± 1,61 і 35,3 ± 1,55 нмоль NADPH/хв на 1мг білка (Р ≤ 0,01) та 1,67 ± 0,041 і 1,64 ± 0,039 нмоль NADPH/хв на 1мг білка (Р ≤ 0,001). Отже, проведені дослідження на бугайцях вказують на те, що кормова добавка “Метісевіт плюс” при згодовуванні бугайцям, за умов навантаження Кадмієм і Свинцем, сприяла активізації глутатіонової системи антиоксидантного захисту за рахунок підвищення рівня ензимної та неензимної її ланок. Проведені нами дослідження підтверджують доцільність застосування кормової добавки “Метісевіт плюс” для профілактики свинцево-кадмієвого токсикозу