Proposed classification of adenomyosis in Infertile women to simplify management options undergoing ART.

OBJECTIVE: Adenomyosis associated with subfertility is a situation of a dilemma for the treating clinician as the treatment is highly controversial and there remains an overall lack of consensus regarding the value of conservative surgery with or without medical management to improve reproductive out-comes. Hence we proposed this classification based on mapping of the size of adenomyoma, its location, distance from the endometrial cavity, and any associated endometriosis by studying 100 women with adenomyosis undergoing IVF. METHODS: We did a prospective study over 2 years in 100 women with adenomyosis who underwent IVF. They were classified into 4 categories based on our management-based proposed classification and the pregnancy outcomes were studied in each group. RESULTS: According to our classification, 56% of women belonged to grade 1, 24% to grade 2, 8% to grade 3, and 12% to Grade 4 Adenomyosis. The Pregnancy rates were 71% in Grade 1, 66% with Medical management, and 33% with surgical management in Grade 2, Grade 3 were offered surrogacy, and 66% in Grade 4 Adenomyosis. CONCLUSIONS: Our classification is simple and allows cost-effective management based on the location and ex-tent of the disease with the help of ultrasonography.

Near infra-red absorbing Quinolizidine fused curcuminoid-BF2 chelate and its applications in photodynamic therapy using MCF-7 and A549 cells.

Metal-free near-infrared absorbing photosensitizers (PS) have been considered promising candidates for photodynamic therapy. Curcumin, curcuminoid, and its derivatives have therapeutic values due to their anti-inflammatory, antifungal, and antiproliferative properties. Curcuminoid-BF2 chelates have also been studied as cell imaging probes, however, their applications in photodynamic therapy are rare. In this article, we describe the synthesis and therapeutic evaluation of quinolizidine fused curcuminoid-BF2 chelate (Quinolizidine CUR-BF2) containing an acid-sensitive group. This donor-acceptor-donor curcuminoid-BF2 derivative exhibits absorption and emission in the deep red region with an absorption band maximum of ∼647 nm and a weak emission band at approximately 713 nm. It is interesting to note that this derivative has a high molar extinction coefficient (164,655 M-1cm-1). Quinolizidine CUR-BF2 possesses intramolecular charge transfer properties, facilitating the production of singlet oxygen (1O2), which plays a crucial role in cell death. Additionally, Quinolizidine CUR-BF2 can enable the selective release of active ingredients in an acidic medium (pH 5). Furthermore, the nanoaggregates of PS were prepared by encapsulating Quinolizidine CUR-BF2 within Pluronic F127 block co-polymer for better water-dispersibility and enhanced cellular uptake. Dark cytotoxicity of nanoaggregates was found to be negligible, whereas they exhibited significant photoinduced cytotoxicity towards cancer cells (MCF-7 and A549) under irradiation of 635 nm light. Further, the cell death pathway using Quinolizidine CUR-BF2 nanoaggregates as PS is found to occur through apoptosis. Specifically, the present study deals with the successful preparation of Quinolizidine CUR-BF2 nanoaggregates for enhanced water-dispersibility and cellular uptake as well as the efficacy evaluation of developed nanoaggregates for photodynamic therapy.

Time-Restricted Eating and Its Metabolic Benefits.

Newer management strategies are being evaluated to treat obesity, which continues to increase worldwide. After 12 h of fasting, the body switches from glucose to fat metabolism, regulating protein synthesis and autophagy. These cellular responses are central to the metabolic benefits of time-restricted eating (TRE), independent of calorie restriction and weight loss, and they have heightened interest in TRE regimens. Controversy remains, however, regarding the benefits of TRE regimens. We reviewed the current literature and concluded that TRE is equivalent to calorie restriction for weight loss and has positive effects for patients with diseases such as nonalcoholic fatty liver disease, cancer, and cardiovascular disease.

PARP1 inhibition protects mice against Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.

Medroxyprogesterone Acetate versus Gonadotropin-Releasing Hormone Antagonist for the Prevention of Premature Luteinizing Hormone Surge in hyper-responder women undergoing controlled ovarian stimulation for IVF/ICSI Cycles.

OBJECTIVE: To compare the effect of Medroxyprogesterone acetate versus Gonadotropin releasing hormone antagonist for the prevention of premature luteinizing hormone (LH) surge in infertile hyper-responder women undergoing controlled ovarian stimulation for in vitro fertilization (IVF) /intracytoplasmic sperm injection (ICSI) cycles. METHODS: One hundred infertile hyper-responder women who were candidate for IVF/ICSI were randomly assigned into two groups. Group 1 was given 20 mg Medroxyprogesterone acetate from day 1 of the menstrual cycle till trigger day. Group 2 was given GnRH antagonist (injection Cetrorelix 0.25 mg s/c) from the day when the leading follicle reached 14 mm until the day of trigger for the prevention of premature LH surge (flexible protocol). We measured LH serum levels on day 1, day 7 of cycle and on trigger day. The primary outcome measured was the incidence of premature LH surge. Other outcome measures were total number of mature follicles on trigger day, total number of mature oocytes retrieved and number of good quality day-3 embryos. RESULTS: There was no premature luteinizing hormone surge in both groups of our study. The mean number of follicles on trigger day, mean number of M2 oocytes retrieved and mean number of good quality day-3 embryos were comparable in both the groups, with no statistically significant difference. CONCLUSIONS: The results of this study stated that MPA can be an effective alternative to GnRH antagonist for the prevention of premature LH surge in hyper-responder women undergoing COS for IVF. It is easy to use, widely available and cost-effective. It may establish a new regimen of ovarian stimulation using MPA as an oral alternative to GnRH antagonist treatment in hyper-responders.

Microscopy-Based Assessment of Fatty Acid Uptake and Lipid Accumulation in Cultured Cells.

The heart relies predominantly on the use of fatty acids to derive energy. Metabolic disorders such as obesity, insulin resistance, and diabetes pose a major risk factor for the development of heart failure. Dysregulation of lipid metabolism observed in these diseases manifests as cardiac lipotoxicity, and is associated with cardiac dysfunction. The alarming rise in the incidence of these metabolic disorders warrants the need for tools to investigate the underlying molecular mechanisms. In this article, we describe a confocal microscopy-based approach to monitor fatty acid uptake and lipid accumulation in vitro, in neonatal murine cardiomyocytes and H9c2 cells. The protocol for assessment of fatty acid uptake relies on the use of BODIPY FL C 12™ to study the kinetics of fatty acid uptake via real-time imaging of fatty acid uptake in live cells. Importantly, it circumvents the need for radioactive labeling of fatty acids to evaluate their uptake. Similarly, the protocol for assessment of lipid accumulation relies on the use of BODIPY™ 493/503 to stain the cytosolic neutral lipid population in fixed cells. We couple these confocal microscopy-based approaches with fluorescence intensity analysis using FIJI to quantify fatty acid uptake and lipid accumulation in vitro. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assessment of fatty acid uptake Basic Protocol 2: Assessment of lipid accumulation.

Sirtuin 6 inhibition protects against glucocorticoid-induced skeletal muscle atrophy by regulating IGF/PI3K/AKT signaling.

Chronic activation of stress hormones such as glucocorticoids leads to skeletal muscle wasting in mammals. However, the molecular events that mediate glucocorticoid-induced muscle wasting are not well understood. Here, we show that SIRT6, a chromatin-associated deacetylase indirectly regulates glucocorticoid-induced muscle wasting by modulating IGF/PI3K/AKT signaling. Our results show that SIRT6 levels are increased during glucocorticoid-induced reduction of myotube size and during skeletal muscle atrophy in mice. Notably, overexpression of SIRT6 spontaneously decreases the size of primary myotubes in a cell-autonomous manner. On the other hand, SIRT6 depletion increases the diameter of myotubes and protects them against glucocorticoid-induced reduction in myotube size, which is associated with enhanced protein synthesis and repression of atrogenes. In line with this, we find that muscle-specific SIRT6 deficient mice are resistant to glucocorticoid-induced muscle wasting. Mechanistically, we find that SIRT6 deficiency hyperactivates IGF/PI3K/AKT signaling through c-Jun transcription factor-mediated increase in IGF2 expression. The increased activation, in turn, leads to nuclear exclusion and transcriptional repression of the FoxO transcription factor, a key activator of muscle atrophy. Further, we find that pharmacological inhibition of SIRT6 protects against glucocorticoid-induced muscle wasting in mice by regulating IGF/PI3K/AKT signaling implicating the role of SIRT6 in glucocorticoid-induced muscle atrophy.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

Role of FoxO transcription factors in aging-associated cardiovascular diseases.

Aging constitutes a major risk factor toward the development of cardiovascular diseases (CVDs). The aging heart undergoes several changes at the molecular, cellular and physiological levels, which diminishes its contractile function and weakens stress tolerance. Further, old age increases the exposure to risk factors such as hypertension, diabetes and hypercholesterolemia. Notably, research in the past decades have identified FoxO subfamily of the forkhead transcription factors as key players in regulating diverse cellular processes linked to cardiac aging and diseases. In the present chapter, we discuss the important role of FoxO in the development of various aging-associated cardiovascular complications such as cardiac hypertrophy, cardiac fibrosis, heart failure, vascular dysfunction, atherosclerosis, hypertension and myocardial ischemia. Besides, we will also discuss the role of FoxO in cardiometabolic alterations, autophagy and proteasomal degradation, which are implicated in aging-associated cardiac dysfunction.

Recapitulating pathophysiology of skeletal muscle diseases in vitro using primary mouse myoblasts on a nanofibrous platform.

Tissue engineering approaches are used to mimic the microenvironment of the skeletal muscle in vitro. However, the validation of a bioengineered muscle as a model to study diseases is inadequate. Here, we present polycaprolactone nanofibers as a robust platform that mimics cellular organization and recapitulates critical functions of the myotubes observed in vivo. We isolated myoblasts from mice following a simplified protocol and cultured them on aligned nanofibers. Myotubes grown on aligned nanofibers maintained alignment for 14 days and exhibited a time-dependent increase in levels of p-AKT upon insulin stimulation. Treatment with matrix-assisted integrin inhibitor led to reduction in p-AKT levels, underscoring the critical role of environment on the biological processes. We demonstrate the suitability of myotubes grown on nanofibrous platform to study corticosteroid-induced muscle degeneration. This study, thus, demonstrates that aligned nanofibers retain myotubes in culture for longer duration and recapitulate the functions of skeletal muscle under pathophysiological conditions.

Successful Application of Combined Autologous Bone Marrow-Derived Stem Cells and Platelet-Rich Plasma in a Case of Severe Asherman Syndrome and Subsequent in vitro Fertilization Conception.

The application of stem cells in infertility is still experimental. Thin endometrium is an important cause of cancelled or less successful frozen embryo transfer cycles. Clinically, numerous strategies have been adopted to promote endometrial regeneration including extended oestrogen administration, low-dose aspirin, pentoxifylline, tocopherol, vaginal sildenafil citrate and intrauterine perfusion with granulocyte colony-stimulating factor. However, even with the use of these therapies, the endometrium in some patients still remains unresponsive. Latest research shows that autologous bone marrow-derived stem cells (ABMDSCs) can be used for regeneration of damaged endometrium. We present a case report of patient with thin endometrium who was successfully treated with the combined use of ABMDSCs mixed with platelet-rich plasma, leading to successful in vitro fertilization conceived pregnancy. Patient consent and due ethical clearance were taken before starting the procedure.

Measuring Protein Synthesis in Cultured Cells and Mouse Tissues Using the Non-radioactive SUnSET Assay.

Changes in protein synthesis occur under diverse physiological and pathological conditions. For example, translation can increase in response to growth signals or decrease in response to pathological states. Such changes have traditionally been measured by tracking the incorporation of radiolabeled amino acids. However, use of radioactivity is increasingly disfavored, and a simple and efficient puromycin-based, non-radioactive method called the SUnSET assay has gained popularity for measuring protein synthesis in diverse cell types and tissues. Here, we describe the principles, procedures, and troubleshooting steps for measuring protein synthesis using the SUnSET assay in cultured cells and mouse tissues. © 2020 Wiley Periodicals LLC Basic Protocol 1: Measuring protein synthesis in cultured cells by western blotting Support Protocol 1: Ponceau staining Support Protocol 2: Testing the specificity of the anti-puromycin antibody Basic Protocol 2: Measuring protein synthesis in cultured cells by immunofluorescence Basic Protocol 3: Measuring protein synthesis in mouse tissues by western blotting.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Toll-like receptor 2 deficiency hyperactivates the FoxO1 transcription factor and induces aging-associated cardiac dysfunction in mice.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors involved in innate immunity. Previous studies have shown that TLR2 inhibition protects the heart from acute stress, including myocardial infarction and doxorubicin-induced cardiotoxicity in animal models. However, the role of TLR2 in the development of aging-associated heart failure is not known. In this work, we studied aging-associated changes in structure and function of TLR2-deficient mice hearts. Whereas young TLR2-KO mice did not develop marked cardiac dysfunction, 8- and 12-month-old TLR2-KO mice exhibited spontaneous adverse cardiac remodeling and cardiac dysfunction in an age-dependent manner. The hearts of the 8-month-old TLR2-KO mice had increased fibrosis, cell death, and reactivation of fetal genes. Moreover, TLR2-KO hearts displayed reduced infiltration by macrophages, increased numbers of myofibroblasts and atrophic cardiomyocytes, and higher levels of the atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Mechanistically, TLR2 deficiency impaired the PI3K/Akt signaling pathway, leading to hyperactivation of the transcription factor Forkhead box protein O1 (FoxO1) and, in turn, to elevated expression of FoxO target genes involved in the regulation of muscle wasting and cell death. AS1842856-mediated chemical inhibition of FoxO1 reduced the expression of the atrophy-related ubiquitin ligases and significantly reversed the adverse cardiac remodeling while improving the contractile functions in the TLR2-KO mice. Interestingly, TLR2 levels decreased in hearts of older mice, and the activation of TLR1/2 signaling improved cardiac functions in these mice. These findings suggest that TLR2 signaling is essential for protecting the heart against aging-associated adverse remodeling and contractile dysfunction in mice.

Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy.

Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.

SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation.

Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3α and GSK3ß are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3ß, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3ß is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity.

SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis.

Heart failure is an aging-associated disease that is the leading cause of death worldwide. Sirtuin family members have been largely studied in the context of aging and aging-associated diseases. Sirtuin 2 (SIRT2) is a cytoplasmic protein in the family of sirtuins that are NAD+-dependent class III histone deacetylases. In this work, we studied the role of SIRT2 in regulating nuclear factor of activated T-cells (NFAT) transcription factor and the development of cardiac hypertrophy. Confocal microscopy analysis indicated that SIRT2 is localized in the cytoplasm of cardiomyocytes and SIRT2 levels are reduced during pathological hypertrophy of the heart. SIRT2-deficient mice develop spontaneous pathological cardiac hypertrophy, remodeling, fibrosis, and dysfunction in an age-dependent manner. Moreover, young SIRT2-deficient mice develop exacerbated agonist-induced hypertrophy. In contrast, SIRT2 overexpression attenuated agonist-induced cardiac hypertrophy in cardiomyocytes in a cell-autonomous manner. Mechanistically, SIRT2 binds to and deacetylates NFATc2 transcription factor. SIRT2 deficiency stabilizes NFATc2 and enhances nuclear localization of NFATc2, resulting in increased transcription activity. Our results suggest that inhibition of NFAT rescues the cardiac dysfunction in SIRT2-deficient mice. Thus, our study establishes SIRT2 as a novel endogenous negative regulator of NFAT transcription factor.

SIRT2 regulates oxidative stress-induced cell death through deacetylation of c-Jun NH2-terminal kinase.

c-Jun NH2-terminal kinases (JNKs) are responsive to stress stimuli and their activation regulate key cellular functions, including cell survival, growth, differentiation and aging. Previous studies demonstrate that activation of JNK requires dual phosphorylation by the mitogen-activated protein kinase kinases. However, other post-translational mechanisms involved in regulating the activity of JNK have been poorly understood. In this work, we studied the functional significance of reversible lysine acetylation in regulating the kinase activity of JNK. We found that the acetyl transferase p300 binds to, acetylates and inhibits kinase activity of JNK. Using tandem mass spectrometry, molecular modelling and molecular dynamics simulations, we found that acetylation of JNK at Lys153 would hinder the stable interactions of the negatively charged phosphates and prevent the adenosine binding to JNK. Our screening for the deacetylases found SIRT2 as a deacetylase for JNK. Mechanistically, SIRT2-dependent deacetylation enhances ATP binding and enzymatic activity of JNK towards c-Jun. Furthermore, SIRT2-mediated deacetylation favours the phosphorylation of JNK by MKK4, an upstream kinase. Our results indicate that deacetylation of JNK by SIRT2 promotes oxidative stress-induced cell death. Conversely, SIRT2 inhibition attenuates H2O2-mediated cell death in HeLa cells. SIRT2-deficient (SIRT2-KO) mice exhibit increased acetylation of JNK, which is associated with markedly reduced catalytic activity of JNK in the liver. Interestingly, SIRT2-KO mice were resistant to acetaminophen-induced liver toxicity. SIRT2-KO mice show lower cell death, minimal degenerative changes, improved liver function and survival following acetaminophen treatment. Overall, our work identifies SIRT2-mediated deacetylation of JNK as a critical regulator of cell survival during oxidative stress.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart.

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.