Formation Of Cyst-like Structures Research Articles

HDAC1/2 and HDAC3 play distinct roles in controlling adult Meibomian gland homeostasis

PurposeTo investigate the roles of HDAC1/2 and HDAC3 in adult Meibomian gland (MG) homeostasis. MethodsHDAC1/2 or HDAC3 were inducibly deleted in MG epithelial cells of adult mice. The morphology of MG was examined. Proliferation, apoptosis, and expression of MG acinus and duct marker genes, meibocyte differentiation genes, and HDAC target genes, were analyzed via immunofluorescence, TUNEL assay, and RNA in situ hybridization. ResultsCo-deletion of HDAC1/2 in MG epithelium caused gradual loss of acini and formation of cyst-like structures in the central duct. These phenotypes required homozygous deletion of both HDAC1 and HDAC2, indicating that they function redundantly in the adult MG. Short-term deletion of HDAC1/2 in MG epithelium had little effect on meibocyte maturation but caused decreased proliferation of acinar basal cells, excessive DNA damage, ectopic apoptosis, and increased p53 acetylation and p16 expression in the MG. By contrast, HDAC3 deletion in MG epithelium caused dilation of central duct, atrophy of acini, defective meibocyte maturation, increased acinar basal cell proliferation, and ectopic apoptosis and DNA damage. Levels of p53 acetylation and p21 expression were elevated in HDAC3-deficient MGs, while the expression of the differentiation regulator PPARγ and the differentiation markers PLIN2 and FASN was downregulated. ConclusionsHDAC1 and HDAC2 function redundantly in adult Meibomian gland epithelial progenitor cells and are essential for their proliferation and survival, but not for acinar differentiation, while HDAC3 is required to limit acinar progenitor cell proliferation and permit differentiation. HDAC1/2 and HDAC3 have partially overlapping roles in maintaining survival of MG cells.

Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro.

The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well understood. Maintaining cells infected with T. gondii type II and III strains under stress conditions induces the tachyzoite-bradyzoite in vitro differentiation, along with the formation of cyst-like structures. However, due to the long exposure to such conditions required to induce the differentiation, the severe damages in the host cell and the low encystation frequency, it has been difficult to dissect in more detail these processes. Here, we successfully induced the in vitro formation of Toxoplasma cysts-like structures from tachyzoites of the type I RH strain by treating with mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase. Mycophenolic acid is a drug widely used for HXGPRT positive selection of Toxoplasma mutant strains along with xanthine incubation in the culture medium; under such conditions, formation of tissue cysts has not been reported. We show that the exposure of extracellular tachyzoites to mycophenolic acid in absence of xanthine, followed by host cell invasion, triggered their differentiation into cyst-like structures. The differential expression of CST1, BAG1, and SAG1 molecules, as well as the structural modifications of infected cells, was characterized during the formation of cyst-like structures in vitro. These findings will allow the characterization of signaling pathways involved in tachyzoite to bradyzoite conversion and formation of tissue cysts.

The aspartic protease napsin A suppresses tumor growth independent of its catalytic activity

Members of the aspartic protease family have been implicated in cancer progression. The aspartic protease napsin A is expressed in type II cells of the lung, where it is involved in the processing of surfactant protein B (SP-B). Napsin A is also expressed in kidney, where its function is unknown. Here, we examined napsin A mRNA expression in human kidney tissues using in situ hybridization. Whereas strong napsin A mRNA expression was observed in kidney proximal tubules, expression was detected in only one of 29 renal cell carcinomas. This result is consistent with previous observations of loss of napsin A expression in high-grade lung adenocarcinomas. We re-expressed napsin A in the tumorigenic HEK293 kidney cell line and examined the phenotype of stably transfected cells. Napsin A-expressing HEK293 cells showed an altered phenotype characterized by formation of cyst-like structures in three-dimensional collagen cultures. Napsin A-expressing cells also showed reduced capacity for anchorage-independent growth and formed tumors in SCID mice with a lower efficiency and slower onset compared to vector-transfected control cells. Mutation of one of the aspartic acid residues in the napsin A catalytic site inactivated enzymatic activity, but did not influence the ability to suppress colony formation in soft agar and tumor formation. The mutation of the catalytic site did not affect processing, glycosylation or intracellular localization of napsin A. These data show that napsin A inhibits tumor growth of HEK293 cells by a mechanism independent of its catalytic activity.

Successful implantation of in vitro-matured, electro-activated oocytes in the pig

In the present study, we derived parthenogenetic porcine fetuses from in vitro-matured oocytes following a simple activation process in order to evaluate their developmental limitations in-vivo. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h. They were subjected to a single square pulse of direct current for 100 μsec at 1,500 V/cm and then treated with 5 μg/mL cytochalasin B for 4 h to obtain activated diploid oocytes. The diploids were cultured in modified Whitten's medium until transfer. Diploids which had cleaved to the 2 and 3 to 4-cell stages were transferred to oviducts of recipients. Live and/or dead parthenogenetic fetuses were recovered in 6 of 8 trials at 17, 18, 19, 24 and 29 d post activation. The total proportion of fetuses to transferred diploids was 31.3% (62/198). When fetuses were recovered at 19 d post activation, the proportion of development into fetuses was 71% (15/21). Our results, however, suggest that periods of gestation longer than 19 d resulted in a decrease of these proportions to 45% (18/40) at 24 d and to 18% (7/40) at 29 d. The hearts were beating in nearly all of the fetuses recovered at 19, 24 and 29 d post activation. Thus, parthenogenetic porcine diploids developed to at least the stage of limb-bud formation beyond the early heart-beating stage. Abnormalities were also externally visible on some fetuses. Formation of cyst-like structures in the heart and liver, and insufficient development of the head region and acephali were observed in some cases.

Unusual in vitro formation of cyst-like structures associated with human intestinal spirochaetosis

Spirochaetes were isolated from rectal biopsies of three patients and successfully cultured. Enzymatic reactions and electron microscopy revealed spirochaetes resembling Brachyspira aalborgi. Examination of ultrathin sections of centrifugates of cultured spirochaetes yielded unusual cyst-like structures with an outer double membrane containing spirochaetes in different developmental stages. The protoplasmic cylinders seemed to originate from a large electron-dense focus in the cyst-like structures. The axial fibrils occurred as loosely distributed solitary structures in the cyst-like structures. Cysts were not found in the biopsy tissue. The encystment of the spirochaetes could be related to their protection, multiplication, spread and transmission.

Formation of cystlike structures by iron-limited Azotobacter vinelandii strain UW during prolonged storage

Slant or liquid cultures of nonencapsulated Azotobacter vinelandii strain UW survived at least 2 years at 20 °C, whereas parallel cultures died after 6 months storage at 4 °C. Survival was decreased at 20 °C when the growth medium was acidic and it was the least at 4 °C when the growth medium was iron limited. Although strain UW was capable of forming cyclopropyl fatty acids and alkyl resorcinols, these indicators of encystment were neither abundant nor present, respectively, in 1.7-year-old cells. Only lauric acid was more abundant in lipid extracts from old cells than from vegetative cells or mature cysts. The old cells were very fragile and rapidly lost viability upon dilution or dehydration. Electron microscopy revealed the production of fragile cystlike structures in iron-limited medium after 1.7 years at 20 °C. These structures had a distinct exine layer composed of membranelike plates, a poorly defined intine, and an extremely dense central body lacking storage granules or internal membranes. Iron-sufficient cultures produced similar but free central bodies, although cystlike structures were rarely seen.

CYSTIC TUBERCULOSIS OF THE BONES IN CHILDREN<subtitle>Report of Two Cases</subtitle>

TUBERCULOSIS of the long bones in children usually attacks the metaphysis, with early caseation and abscess formation. Less commonly, single or multiple tuberculous foci are found in the diaphyses, with localized thinning of the cortex and formation of cyst-like structures. These tuberculous cysts may remain stationary for long periods, may progress to abscess formation, or, more frequently, may heal without complication. Such cyst-like areas in roentgenograms of the long bones, especially when unaccompanied by sinus or abscess formation, may present problems in differential diagnosis. For this reason, we present the following two cases of cystic tuberculosis of the bones. REPORT OF CASES Case1.–B. T., an 18-month-old Negro girl, was admitted to the Pediatric Clinic of Provident Hospital (Chicago) on Jan. 27, 1938, with the complaint of disability in the region of the left shoulder. Past History .–The child was born at term of a normal spontaneous delivery. The birth