Intestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune ...response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.
Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.
Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.
Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.
•Current literature suggests comparable Ti and PEEK cage spinal fusion rates.•Of the 2 cage materials, Ti is associated with increased subsidence.•Prospective studies should identify the benefits of ...one material over the other.
Spinal interbody fusion is a standard and accepted method for spinal fusion. Interbody fusion devices include titanium (Ti) and polyetheretherketone (PEEK) cages with distinct biomechanical properties. Titanium and PEEK cages have been evaluated in the cervical and lumbar spine, with conflicting results in bony fusion and subsidence. Using Preferred Reporting Items for Systematic reviews and Meta-analyses (PRISMA) guidelines, we reviewed the available literature evaluating Ti and PEEK cages to assess subsidence and fusion rates. Six studies were included in the analysis, 3 of which were class IV evidence, 2 were class III, and 1 was class II. A total of 410 patients (Ti-228, PEEK-182) and 587 levels (Ti-327, PEEK-260) were studied. Pooled mean age was 50.8years in the Ti group, and 53.1years in the PEEK group. Anterior cervical discectomy was performed in 4 studies (395 levels) and transforaminal interbody fusion in 2 studies (192 levels). No statistically significant difference was found between groups with fusion (OR 1.16, 95% C.I 0.59–2.89, p=0.686, I2=49.7%) but there was a statistically significant the rate of subsidence with titanium (OR 3.59, 95% C.I 1.28–10.07, p=0.015, I2=56.9%) at last follow-up. Titanium and PEEK cages are associated with a similar rate of fusion, but there is an increased rate of subsidence with titanium cage. Future prospective randomized controlled trials are needed to further evaluate these cages using surgical and patient-reported outcomes.
Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the ...chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV ...transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
The anterior bundle of the ulnar collateral ligament (UCL) is the primary anatomical structure providing elbow stability in overhead sports, particularly baseball. Injury to the UCL in ...overhead athletes often leads to symptomatic valgus instability that requires surgical treatment.
Hypothesis
Ulnar collateral ligament reconstruction with a free tendon graft, known as Tommy John surgery, will allow return to the same competitive level of sports participation in the majority of athletes.
Study Design
Case series; Level of evidence, 4.
Methods
Ulnar collateral reconstruction (1266) or repair (15) was performed in 1281 patients over a 19-year period (1988—2006) using a modification of the Jobe technique. Data were collected prospectively and patients were surveyed retrospectively with a telephone questionnaire to determine outcomes and return to performance at a minimum of 2 years after surgery.
Results
Nine hundred forty-two patients were available for a minimum 2-year follow-up (average, 38.4 months; range, 24-130 months). Seven hundred forty-three patients (79%) were contacted for follow-up evaluation and/or completed a questionnaire at an average of 37 months postoperatively. Six hundred seventeen patients (83%) returned to the previous level of competition or higher, including 610 (83%) after reconstruction. The average time from surgery to the initiation of throwing was 4.4 months (range, 2.8-12 months) and the average time to full competition was 11.6 months (range, 3-72 months) after reconstruction. Complications occurred in 148 patients (20%), including 16% considered minor and 4% considered major.
Conclusion
Ulnar collateral ligament reconstruction with subcutaneous ulnar nerve transposition was found to be effective in correcting valgus elbow instability in the overhead athlete and allowed most athletes (83%) to return to previous or higher level of competition in less than 1 year.
Here, we provide an overview of the different mechanisms whereby three different chaperones, Spy, Hsp70, and Hsp60, interact with folding proteins, and we discuss how these chaperones may guide the ...folding process. Available evidence suggests that even a single chaperone can use many mechanisms to aid in protein folding, most likely due to the need for most chaperones to bind clients promiscuously. Chaperone mechanism may be better understood by always considering it in the context of the client's folding pathway and biological function.
In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ...ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.
Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and ...genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.
A machine learning framework is developed to estimate ocean-wave conditions. By supervised training of machine learning models on many thousands of iterations of a physics-based wave model, accurate ...representations of significant wave heights and period can be used to predict ocean conditions. A model of Monterey Bay was used as the example test site; it was forced by measured wave conditions, ocean-current nowcasts, and reported winds. These input data along with model outputs of spatially variable wave heights and characteristic period were aggregated into supervised learning training and test data sets, which were supplied to machine learning models. These machine learning models replicated wave heights from the physics-based model with a root-mean-squared error of 9 cm and correctly identify over 90% of the characteristic periods for the test-data sets. Impressively, transforming model inputs to outputs through matrix operations requires only a fraction (<1/1,000th) of the computation time compared to forecasting with the physics-based model.
•Machine learning models were trained to act as a surrogate for the physics-based SWAN model.•An MLP model accurately represented the significant wave height field and an SVM model simulated the characteristic period.•Machine learning models represented the SWAN-simulated wave conditions in less than 1/1000th of the computational time.
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