Electrochemical Analysis of the Thermal Stability of 0.9-4.1 nm Diameter Gold Nanoclusters.

Here we report the thermal properties of weakly stabilized 0.9, 1.6, and 4.1 nm Au nanoparticles (NPs)/nanoclusters (NCs) attached to indium-tin-oxide- or fluorine-doped-tin-oxide-coated glass electrodes (glass/ITO or glass/FTO). The peak oxidation potential (Ep) for Au measured by anodic stripping voltammetry (ASV) is indicative of the NP/NC size. Heating leads to a positive shift in Ep due to an increase in NP/NC size from thermal ripening. The size transition temperature (Tt) decreases with decreasing NP/NC size following the order of 4.1 nm (509 °C) > 1.6 nm (132 °C) > 0.9 nm (90 °C/109 °C, two transitions) as compared to the bulk melting point (Tm,b) for Au of 1064 °C. The Tt generally agrees with models describing the size-dependent melting point of Au NPs (Tm,NP) for 4.1 and 1.6 nm diameter Au NPs but is higher than the models for 0.9 nm Au NCs. Scanning electron microscopy (SEM) and UV-vis size analysis confirm the electrochemical results. The thermal stability of electrode-supported metal NPs/NCs is important for their effective use in catalysis, sensing, nanoelectronics, photovoltaics, and other applications.

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

Nanoporous Thin Films Formed from Photocleavable Diblock Copolymers on Gold Substrates Modified with Thiolate Self-Assembled Monolayers.

Nanoporous thin films formed on electrodes are considered functional elements of electrochemical sensing systems, thus motivating methods for their development. We report a preparative strategy detailing the effects of surface modification of gold substrates with thiolate self-assembled monolayers (SAMs) on the properties of nanoporous thin films derived from polystyrene-block-poly(ethylene oxide) having a photocleavable o-nitrobenzyl ester junction (PS-hν-PEO). Two PS-hν-PEO having similar PEO volume fractions (≈0.2) but different molecular weights (10 and 23 kg/mol) were used to prepare films (30-100 nm thick) spin-cast on gold substrates unmodified and modified with cysteamine, thioctic acid, and 6-hydroxy-1-hexanethiol SAMs. Solvent vapor annealing followed by PEO removal led to the formation of nanopores with average diameters of 12 and 19 nm from the smaller and larger PS-hν-PEO, respectively. Cyclic voltammograms of 1,1'-ferrocenedimethanol showed that nanoporous films on cysteamine SAMs afforded nanopores reaching the underlying substrates at higher density than those on the other substrates. This result was attributed to balanced affinity of the cysteamine SAM surface with PS and PEO, which enhanced the vertical orientation of PEO microdomains. The generation of carboxyl groups associated with the photocleavage reaction was revealed by pH-dependent changes in the voltammogram of Fe(CN)63- that reflected electrostatic effects regulated by the protonation state of the carboxyl groups. The SAMs underneath the nanoporous films could be replaced by treatment with a thiol solution, as verified by voltammograms of l-ascorbic acid. These results suggest that thiolate SAM modification provides a simple means to control the interfacial orientation of PEO microdomains in thin PS-hν-PEO films.

Inhibition of sonic hedgehog and PI3K/Akt/mTOR pathways cooperate in suppressing survival, self-renewal and tumorigenic potential of glioblastoma-initiating cells.

Since PI3K/Akt/mTOR and sonic hedgehog (SHH) signaling pathways are highly activated in glioblastoma-initiating cells (GICs), we examined the effects of inhibiting these pathways on GIC characteristics and tumor growth in mice. NVP-LDE-225 (inhibitor of Smoothened) inhibited the expression of Gli1, Gli2, Smoothened, Patched1, and Patched2, and induced the expression of SuFu, whereas NVP-BEZ-235 (dual inhibitor of PI3K and mTOR) inhibited the expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting the self-renewal capacity of GICs, expression of pluripotency maintaining factors (Nanog, c-Myc, Oct4, and Sox2), Musashi1, cyclin D1, and Bcl-2, and transcription and expression of Gli, and in inducing the expression of cleaved caspase-3, cleaved PARP and Bim. Additionally, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting epithelial-mesenchymal transition. Finally, the combination of NVP-LDE-225 and NVP-BEZ-235 was superior in inhibiting tumor growth, regulating the expression of pluripotency promoting factors, stem cell markers, cell cycle, and cell proliferation, and modulating EMT compared to single agent alone. In conclusion, the combined inhibition of PI3K/Akt/mTOR and SHH pathways was superior to single pathway inhibition in suppressing glioblastoma growth by targeting GICs.

Size Determination of Metal Nanoparticles Based on Electrochemically Measured Surface-Area-to-Volume Ratios.

Here we report the electrochemical determination of the surface-area-to-volume ratio (SA/ V) of Au nanospheres (NSs) attached to electrode surfaces for size analysis. The SA is determined by electrochemically measuring the number of coulombs of charge passed during the reduction of surface Au2O3 following Au NS oxidation in HClO4, whereas V is determined by electrochemically measuring the coulombs of charge passed during the complete oxidative dissolution of all of the Au in the Au NSs in the presence of Br- to form aqueous soluble AuBr4-. Assuming a spherical geometry and taking into account the total number of Au NSs on the electrode surface, the SA/ V is theoretically equal to 3/radius. A plot of the electrochemically measured SA/ V versus 1/radius for five different-sized Au NSs is linear with a slope of 1.8 instead of the expected value of 3. Following attachment of the Au NSs to the electrode and ozone treatment, the plot of SA/ V versus 1/radius is linear with a slope of 3.5, and the size based on electrochemistry matches very closely with those measured by scanning electron microscopy. We believe the ozone cleans the Au NS surface, allowing a more accurate measurement of the SA.

Aggregation-Dependent Oxidation of Metal Nanoparticles.

Here we describe the effect of aggregation on the oxidation of citrate-stabilized Au nanoparticles (NPs) attached electrostatically to amine-functionalized glass/ITO electrodes. When the Au NPs are attached to the electrode from a solution with pH greater than ∼3.0, they are well-separated on the electrode and oxidize in bromide-containing electrolyte at 0.698, 0.757, and 0.943 V (vs Ag/AgCl) for 4, 15, and 50 nm diameter Au NPs, respectively, in line with their size-dependent oxidation behavior. In solutions below pH 3.0, the Au NPs aggregate in solution and attach to the electrode in the aggregated form. The solution UV-vis spectra and scanning electron microscopy images of the electrodes show clear evidence of aggregation. The oxidation potential for aggregated 4 and 15 nm diameter Au NPs shifts positive by a maximum of 230 and 180 mV, respectively. The magnitude of the shift depends on the extent of aggregation, which was controlled by the solution pH and time. NP aggregation leads to a significant reduction in the surface area-to-volume ratio, which is likely responsible for the positive shift in the oxidation potential. The oxidation potential does not shift at all for aggregated 50 nm diameter Au NPs.

TGFß responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness.

OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.

Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins.

Dipeptidyl-peptidase IV (DPP-IV) inhibitor delays tolerance to anxiolytic effect of ethanol and withdrawal-induced anxiety in rats.

Dipeptidyl-peptidase IV (DPP-IV) is an enzyme responsible for the metabolism of endogenous gut-derived hormone, glucagon-like peptide-1 (GLP-1). DPP-IV is known for its role in energy homeostasis and pharmacological blockade of this enzyme is a recently approved clinical strategy for the management of type II diabetes. Accumulating evidences suggest that enzyme DPP-IV can affect spectrum of central nervous system (CNS) functions. However, little is known about the role of this enzyme in ethanol-mediated neurobehavioral complications. The objective of the present study was to examine the impact of DPP-IV inhibitor, sitagliptin on the development of tolerance to anxiolytic effect of ethanol and anxiety associated with ethanol withdrawal in rats. A dose-response study revealed that sitaglitpin (20 mg/kg, p.o.) per se exhibit anxiolytic effect in the elevated plus maze (EPM) test in rats. Tolerance to anxiolytic effect of ethanol (2 g/kg, i.p.; 8 % w/v) was observed from 7(th) day of ethanol-diet (6 % v/v) consumption. In contrast, tolerance to anxiolytic effect of ethanol was delayed in rats that were treated daily with sitagliptin (20 mg/kg, p.o.) as tolerance was observed from 13(th)day since commencement of ethanol-diet consumption. Discontinuation of rats from ethanol-diet after 15-days of ethanol consumption resulted in withdrawal anxiety between 8 h and 12 h post-abstinence. However, rats on 15-day ethanol-diet with concomitant sitagliptin (20 mg/kg, p.o.) treatment exhibited delay in appearance (24 h post-withdrawal) of withdrawal anxiety. In summary, DPP-IV inhibitors may prove as an attractive research strategy against ethanol tolerance and dependence.

Glucagon-like peptide-1 (GLP-1) receptor agonist prevents development of tolerance to anti-anxiety effect of ethanol and withdrawal-induced anxiety in rats.

UNLABELLED: Despite major advances in the understanding about ethanol actions, the precise underlying neurobiological mechanisms for ethanol dependence remain largely elusive. We recently reported that inhibition of dipeptidyl-peptidase IV (DPP-IV), an enzyme responsible for metabolism of endogenous glucagon-like peptide-1 (GLP-1), delays tolerance to anti-anxiety effect of ethanol and withdrawal-induced anxiety in rats. Intrigued with this report, present study examined the role of glucagon-like peptide-1 (GLP-1) receptor agonist, liraglutide in (1) acute anti-anxiety effect of ethanol; (2) tolerance to ethanol's anti-anxiety-effect and (3) ethanol withdrawal-induced anxiety using elevated plus maze (EPM) test in rats. Ethanol (2 g/kg, i.p.; 8 % w/v) and liraglutide (50 µg/kg, i.p.) treatments exhibited anti-anxiety effect in EPM test. Doses of ethanol (1.0 or 1.5 g/kg, i.p.) that were not effective per se elicited anti-anxiety when combined with sub-effective dose of liraglutide (25 µg/kg, i.p.). Rats consuming ethanol-diet (6 % v/v) exhibited tolerance to anti-anxiety effect of ethanol from day-7 of ethanol consumption. Peak ethanol withdrawal-induced anxiety was observed at 8-10 h upon abstinence from ethanol-diet after 15-days consumption. Rats on simultaneous once-daily liraglutide treatment (50 µg/kg, i.p.) neither had any signs of tolerance to anti-anxiety effect of ethanol nor did they exhibit withdrawal-induced anxiety. IN CONCLUSION: (1) GLP-1 agonist, liraglutide exhibited anti-anxiety effect per se; (2) potentiated anti-anxiety effect of ethanol; (3) prevented development tolerance to anti-anxiety effect of ethanol and (4) prevented withdrawal-induced anxiety. Further studies examining intracellular cascade of events contributing to these effects may help to improve understanding about role of GLP-1 receptors in ethanol mediated behaviors.

GLP-1 receptor agonist liraglutide reverses long-term atypical antipsychotic treatment associated behavioral depression and metabolic abnormalities in rats.

Mood disorder patients that are on long-term atypical antipsychotics treatment frequently experience metabolic dysfunctions. In addition to this, accumulating evidences points to increased risk of structural abnormalities, brain volume changes, altered neuroplasticity and behavioral depression with long-term antipsychotics use. However, there is paucity of preclinical evidences for long-term antipsychotic associated depression-like behavior. The objectives of the present study were: (1) to evaluate influence of long-term antipsychotic (olanzapine) treatment on rat behavior in forced swim test (FST) as a model for depression and; (2) to examine impact of glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide - an antidiabetic medication for type II diabetes, on long-term olanzapine associated metabolic and behavioral changes in rats. Daily olanzapine treatment (0.5 mg/kg; p.o.) for 8-9 weeks significantly increased body weights, food and water intake, plasma cholesterol and triglycerides and immobility time in FST with parallel reduction in plasma HDL cholesterol levels. These results points to development of metabolic abnormalities and depression-like behavior with long-term olanzapine treatment. Acute liraglutide (50 µg/kg; i.p.) and imipramine (10 mg/kg, i. p.) treatment per se significantly decreased duration of immobility in FST compared to vehicle treated rats. Additionally, 3-week liraglutide treatment (50 µg/kg; i.p., daily) partially reversed metabolic abnormalities and depression-like behavior with long-term olanzapine-treatment in rats. None of these treatment regimens affected locomotor behavior of rats. In summary, add-on GLP-1 receptor agonists promise novel alternatives to counteract long-term antipsychotics associated behavioral and metabolic complications.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC. METHODS: Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay. RESULTS: Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability. CONCLUSION: PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

Vaginal Hysterectomy by Electrosurgery for Benign Indications Associated with Previous Cesarean Section.

Background: Vaginal hysterectomies have been associated with difficulties in patients who have had cesarean sections prior to such hysterectomies. However, the Purohit technique may obviate the problems and make it easier to perform these operations. Objectives: This research tested an approach designed to facilitate vaginal hysterectomy in patients with previous cesarean sections in the absence of fixed adhesions of uterine corpi to previous anterior abdominal scarring. Design/Method: An observational study was conducted in a private general hospital between February 2010 and June 2012. All candidates for hysterectomy for benign indications who had had previous cesarean sections were examined for the presence of clinical and sonographic signs of fixed adhesions of uterine corpi to anterior abdominal-wall incisions caused by prior cesarean sections. Candidates who had such adhesions were not given vaginal hysterectomies. Results: Sixty-four (64) consecutive candidates were selected for vaginal hysterectomies. Of these patients, 26 (40.62%) had 1 cesarean section, 33 (51.56 %) had 2 cesarean sections, and 5 (7.81%) had 3 cesarean sections. Four (4) patients had had prior pelvic operations. The uteri were smaller than 12 weeks' gestation-size in 62 (96.87%) cases. In 26 (40.62 %) cases, there were no obstruction to accessing the anterior cul-de-sacs and vaginal hysterectomies were performed using the Purohit technique. In 38 (59.37 %) cases, dense uterovesical adhesions obstructed access to the anterior cul-de-sacs and a posterioanterior approach was used to perform vaginal hysterectomy in these patients. Vaginal hysterectomy was completed in all 64 cases. Vaginal salpingo-oophorectomy was performed in 3 (4.68%) cases. The mean operative time was 78.59±33.15 (35-190) minutes. The mean weight of specimen uteri was 161.01±108.87 (50-550) g. No patients needed conversions or blood transfusions. No patients had bladder, ureteric, or thermal injuries. Finally, there were no other major postoperative complications. Conclusions: In the absence of fixed adhesions of the uterine corpus to previous anterior abdominal scarring, vaginal hysterectomy for benign indications associated with previous cesarean section may be accomplished safely. The posterioanterior approach during vaginal hysterectomy may avoid unintended bladder injury in the presence of dense uterovesical adhesions caused by previous cesarean sections. (J GYNECOL SURG 29:7).

A Vagino-Laparoscopic Strategy for Hysterectomy of Large Uteri to Ease Surgical Challenges.

Purpose: To develop a vagino-laparoscopic strategy for hysterectomy (VLH) to treat uteri with large fibroids at ≥ 16 weeks of gestation to ease surgical challenges. Patients and Methods: This was an observational study conducted in our private general hospital on 64 consecutive cases involving uteri with large and benign fibroids at ≥ 16 weeks of gestation. We excluded cases with an associated ventro-fixed uterus and large cervical fibroids. Interventions: The VLH strategy involves vaginal separation of the uterosacral with the uterine arteries followed by laparoscopic separation of the remaining upper pedicles. The uterus was then removed vaginally. Results: The largest uteri receiving treatment was at 26 weeks of gestation and the mean uterus weight was 869.60 ± 275.10 g (range: 500-1900 g). The VLH strategy was successful in 63 (98.43%) cases irrespective of the configuration of the uterus. One case (1.56%) required mini-laparotomy conversion due to the need for adhesiolysis; this case exhibited extensive adhesion of the rectum to the posterior wall of the uterus due to a history of previous myomectomy of the posterior wall. There were no conversions due to failed laparoscopic exposure of the adnexal or uterine vascular pedicles, or due to uncontrolled intraoperative bleeding. There were no cases of urinary tract injury or other major complications. The vaginal detachment of uterosacral ligaments before the laparoscopic phase of the VLH strategy increased upwards mobility and dislodged the cervico-isthmic level of large uteri from the narrow lower aspect of the pelvis to the upper wider part of the pelvis; this eased the surgical challenges associated with such cases and avoided complications during laparoscopy. Conclusion: We developed a vagino-laparoscopic strategy for hysterectomy that can ease the technical challenges associated with the majority of large uteri.

Vagino-Laparoscopic Conservative Strategy of Hysterectomy in Indicated Cases of Severe Pelvic Endometriosis Followed by 24 Months of Depot-Medroxyprogesterone Acetate Therapy- A Symptom-Solving Treatment Model to Ease Surgical Challenges.

Purpose: To demonstrate the long-term outcome of a symptom-solving treatment model (SSTM). Patients and Methods: An observational study was carried out between June 2016 and December 2022 in our private setup on consecutive candidates of hysterectomy for severe pelvic endometriosis. Candidates were treated by the SSTM, which constitutes a systematic vagino-laparoscopic conservative strategy of hysterectomy with preservation of the ovary or ovaries followed by 24 months of postoperative depot-medroxyprogesterone acetate (DMPA) therapy. Cases were followed up to December 2022, 2.5 years beyond the last hysterectomy in May 2020. Main Outcome Measures: Relief of endometriosis-associated symptoms and prevention of recurrence in the long term. Results: Symptomatic relief of endometriosis-related pain, such as cyclical dysmenorrhoea, pelvic pain, dyschezia, and vaginal pain, occurred in all 68 (100%) cases from the next expected date of menstruation. None of the cases showed a recurrence of endometriosis-related pelvic pain; overall, 37 (54.41%) cases crossed 4-6 years, and 31 (45.58%) cases crossed 2.5-4.0 years following the hysterectomy operation. Four (5.88%) cases had non-endometriotic pelvic pain. None of the cases required repeat surgery or had any major side effects or complications due to DMPA. No major perioperative complications were observed. The results were achieved without the requirement of challenging extensive retroperitoneal laparoscopic dissection, ureterolysis, and rectum surgeries. Conclusion: This SSTM can be an option in indicated cases of severe pelvic endometriosis to provide symptom relief and prevent the recurrence of endometriosis-associated pelvic pain in the long term.

Rescue of Alzheimer's disease phenotype in a mouse model by transplantation of wild-type hematopoietic stem and progenitor cells.

Alzheimer's disease (AD) is the most prevalent cause of dementia; microglia have been implicated in AD pathogenesis, but their role is still matter of debate. Our study showed that single systemic wild-type (WT) hematopoietic stem and progenitor cell (HSPC) transplantation rescued the AD phenotype in 5xFAD mice and that transplantation may prevent microglia activation. Indeed, complete prevention of memory loss and neurocognitive impairment and decrease of ß-amyloid plaques in the hippocampus and cortex were observed in the WT HSPC-transplanted 5xFAD mice compared with untreated 5xFAD mice and with mice transplanted with 5xFAD HSPCs. Neuroinflammation was also significantly reduced. Transcriptomic analysis revealed a significant decrease in gene expression related to "disease-associated microglia" in the cortex and "neurodegeneration-associated endothelial cells" in the hippocampus of the WT HSPC-transplanted 5xFAD mice compared with diseased controls. This work shows that HSPC transplant has the potential to prevent AD-associated complications and represents a promising therapeutic avenue for this disease.

Localized colonic stem cell transplantation enhances tissue regeneration in murine colitis.

Many patients suffer from chronic gastrointestinal diseases characterized by chronic inflammation, increased intestinal permeability and visceral pain in which there is no definitive treatment. Adult stem cells have recently been used in various disease states to contribute wound-healing processes. In the current study we investigated the ability of intra-colonic adult stem cells application to heal colonic inflammation in IL-10(-/-) mice with active colitis. The aims of this study were to determine whether intra-colonic infusion of adult colonic stem cells (CSCs) (local stem cell transplantation): (i) restores intestinal permeability; (ii) attenuates visceral hypersensitivity; (iii) heals murine colitis. IL-10(-/-) mice with active colitis were transplanted with adult stem cells. Mice received either a single intracolonic infusion of CSCs or colonic epithelial cells. Two weeks after transplantation, we measured visceral hypersensitivity and intestinal permeability and correlated these with histological improvement of colitis. IL-10(-/-) mice that received stem cell transplantation showed histopathologic evidence of recovery from colitis. Improvement in colitis as graded by pathology scores correlated with restoration of intestinal permeability and decreased visceral hypersensitivity. Intra-colonic administration of CSCs is a potential therapeutic method for treating refractory symptoms in patients with chronic gastrointestinal diseases associated with chronic inflammation and visceral hypersensitivity. This method may be safer and should have far fewer side effects than systemic stem cell administration.

Functional analysis of microRNAs in human hepatocellular cancer stem cells.

MicroRNAs are endogenous small non-coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self-renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL-6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL-6 and Twist. We demonstrated that conserved let-7 and miR-181 family members were up-regulated in HSCs by global microarray-based microRNA profiling followed by validation with real-time polymerase chain reaction. Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion. Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let-7 directly targets SOCS-1 and caspase-3, whereas miR-181 directly targets RASSF1A, TIMP3 as well as nemo-like kinase (NLK). In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let-7/miR-181s suggesting that let-7 and miR-181 may be molecular targets for eradication of hepatocellular malignancies.

Longitudinal analysis reveals age-related changes in the T cell receptor repertoire of human T cell subsets.

A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRß repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.

CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin (FXN) gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes. In this study, we report the first step toward an autologous HSPC transplantation using the CRISPR-Cas9 system for FRDA. We first identified a pair of CRISPR RNAs (crRNAs) that efficiently removes the GAA expansions in human FRDA lymphoblasts, restoring the non-pathologic level of frataxin expression and normalizing mitochondrial activity. We also optimized the gene-editing approach in HSPCs isolated from healthy and FRDA patients' peripheral blood and demonstrated normal hematopoiesis of gene-edited cells in vitro and in vivo. The procedure did not induce cellular toxic effect or major off-target events, but a p53-mediated cell proliferation delay was observed in the gene-edited cells. This study provides the foundation for the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA.