Faecal Microbiota of Cats with Insulin-Treated Diabetes Mellitus

Microorganisms within the gastrointestinal tract significantly influence metabolic processes within their mammalian host, and recently several groups have sought to characterise the gastrointestinal microbiota of individuals affected by metabolic disease. Differences in the composition of the gastrointestinal microbiota have been reported in mouse models of type 2 diabetes mellitus, as well as in human patients. Diabetes mellitus in cats has many similarities to type 2 diabetes in humans. No studies of the gastrointestinal microbiota of diabetic cats have been previously published. The objectives of this study were to compare the composition of the faecal microbiota of diabetic and non-diabetic cats, and secondarily to determine if host signalment and dietary factors influence the composition of the faecal microbiota in cats. Faecal samples were collected from insulin-treated diabetic and non-diabetic cats, and Illumina sequencing of the 16S rRNA gene and quantitative PCR were performed on each sample. ANOSIM based on the unweighted UniFrac distance metric identified no difference in the composition of the faecal microbiota between diabetic and non-diabetic cats, and no significant differences in the proportions of dominant bacteria by phylum, class, order, family or genus as determined by 16S rRNA gene sequencing were identified between diabetic and non-diabetic cats. qPCR identified a decrease in Faecalibacterium spp. in cats aged over ten years. Cat breed or gender, dietary carbohydrate, protein or fat content, and dietary formulation (wet versus dry food) did not affect the composition of the faecal microbiota. In conclusion, the composition of the faecal microbiota was not altered by the presence of diabetes mellitus in cats. Additional studies that compare the functional products of the microbiota in diabetic and non-diabetic cats are warranted to further investigate the potential impact of the gastrointestinal microbiota on metabolic diseases such as diabetes mellitus in cats.     

mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility

The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.     

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.Dysferlinopathy is a progressive muscle wasting disease, which is classified as limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1, based on its muscle involvement.^{1, 2} Dysferlin deficit leads to altered vesicle formation and trafficking,^{3, 4} poor repair of injured cell membranes,^{5, 6} and increased muscle inflammation.^{7, 8} Dysferlin contains C2 domains that are found in Ca²⁺-dependent membrane fusion proteins such as synaptotagmins.⁹ Thus, dysferlin is thought to regulate muscle function by regulating vesicle trafficking and fusion.^{10, 11, 12, 13} Dysferlin deficiency has also been implicated in conflicting reports regarding the fusion ability of dysferlinopathic myoblasts.^{4, 14, 15, 16} With such diverse roles for dysferlin, the mechanism through which dysferlin deficiency results in muscle pathology is unresolved. As skeletal muscle-specific re-expression of dysferlin rescues all dysferlinopathic pathologies,^{17, 18} myofiber repair has been suggested to be the unifying deficit underlying muscle pathology in dysferlinopathy.¹⁹ Repair of injured cell membranes requires subcellular compartments, which in mammalian cells include lysosomes,¹¹ enlargeosomes,²⁰ caveolae,²¹ dysferlin-containing vesicles,⁵ and mitochondria.²²Cells from muscular dystrophy patients that have normal dysferlin expression exhibit normal lysosome and enlargeosome exocytosis.²³ However, dysferlinopathic muscle cells exhibit enlarged LAMP2-positive lysosomes, reduced fusion of early endosomes, altered expression of proteins regulating late endosome/lysosome fusion, and reduced injury-triggered cell-surface levels of LAMP1.^{4, 11, 12} In non-muscle cells, lack of dysferlin reduces lysosomal exocytosis.²⁴ These findings implicate lysosomes in dysferlin-mediated muscle cell membrane repair. In one model for lysosome-mediated cell membrane repair, Ca²⁺ triggers vesicle–vesicle fusion near the site of injury, forming ‘membrane patch'', which fuses to repair the wounded cell membrane.^{25, 26, 27, 28} In another model, lysosome exocytosis following cell membrane injury by pore-forming toxins leads to secretion of the lysosomal enzyme acid sphingomyelinase (ASM), which causes endocytosis of pores in the damaged cell membranes.^{21, 29, 30} Both these models have been suggested to be involved in the repair of injured muscle cells.^{21, 28}To examine the muscle cell pathology in dysferlinopathy, we have developed dysferlinopathic mouse and human models. Use of these models shows that a lack of dysferlin does not alter myogenic differentiation but causes poor repair of even undifferentiated muscle cells. We show that dysferlin is required for tethering lysosomes to the cell membrane. Fewer lysosomes at the cell membrane in dysferlinopathic cells results in slow and reduced lysosome exocytosis following injury. This reduction in exocytosis reduces injury-triggered ASM secretion, which is responsible for the poor repair of dysferlinopathic muscle cells. Extracellular sphingomyelinase (SM) fully rescues the repair deficit in dysferlinopathic cells and mouse myofibers, offering a potential drug-based therapy for dysferlinopathy.     

Molecular docking analysis of compounds from Andrographis paniculata with EGFR

EGFR is linked with oral cancer. Therefore, it is of interest document the molecular docking analysis of compounds from Andrographis paniculata with EGFR. Data shows the binding features of five compounds 14- acetylandrographolide, andrograpanin, andrographolide, isoandrographolide and neoandrographolide from Andrographis paniculata with EGFR for further consideration.     

Seasonal Variations of Neuromotor Development By 14 Months of Age: Hamamatsu Birth Cohort for Mothers and Children (HBC Study)

The present study aimed at investigating whether neuromotor development, from birth to 14 months of age, shows seasonal, cyclic patterns in association with months of birth. Study participants were 742 infants enrolled in the Hamamatsu Birth Cohort (HBC) Study and followed-up from birth to the 14th month of age. Gross motor skills were assessed at the ages of 6, 10, and 14 months, using Mullen Scales of Early Learning. The score at each assessment was regressed onto a trigonometric function of months of birth, with an adjustment for potential confounders. Gross motor scores at the 6th and 10th months showed significant 1-year-cycle variations, peaking among March- and April-born infants, and among February-born infants, respectively. Changes in gross motor scores between the 10th and 14th months also showed a cyclic variation, peaking among July- and August-born infants. Due to this complementary effect, gross motor scores at the 14th month did not show seasonality. Neuromotor development showed cyclic seasonality during the first year of life. The effects brought about by month of birth disappeared around 1 year of age, and warmer months seemed to accelerate the neuromotor development.     

Platelets Induce Apoptosis during Sepsis in a Contact-Dependent Manner That Is Inhibited by GPIIb/IIIa Blockade

Purpose

End-organ apoptosis is well-described in progressive sepsis and Multiple Organ Dysfunction Syndrome (MODS), especially where platelets accumulate (e.g. spleen and lung). We previously reported an acute sepsis-induced cytotoxic platelet phenotype expressing serine protease granzyme B. We now aim to define the site(s) of and mechanism(s) by which platelet granzyme B induces end-organ apoptosis in sepsis.

Methods

End-organ apoptosis in murine sepsis (i.e. polymicrobial peritonitis) was analyzed by immunohistochemistry. Platelet cytotoxicity was measured by flow cytometry following 90 minute ex vivo co-incubation with healthy murine splenocytes. Sepsis progression was measured via validated preclinical murine sepsis score.

Measurements and Main Results

There was evident apoptosis in spleen, lung, and kidney sections from septic wild type mice. In contrast, there was a lack of TUNEL staining in spleens and lungs from septic granzyme B null mice and these mice survived longer following induction of sepsis than wild type mice. In co-incubation experiments, physical separation of septic platelets from splenocytes by a semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from negative controls. Chemical separation by the platelet GPIIb/IIIa receptor inhibitor eptifibatide decreased apoptosis by 66.6±10.6% (p = 0.008). Mice treated with eptifibatide in vivo survived longer following induction of sepsis than vehicle control mice.

Conclusions

In sepsis, platelet granzyme B-mediated apoptosis occurs in spleen and lung, and absence of granzyme B slows sepsis progression. This process proceeds in a contact-dependent manner that is inhibited ex vivo and in vivo by the platelet GPIIb/IIIa receptor inhibitor eptifibatide. The GPIIb/IIIa inhibitors and other classes of anti-platelet drugs may be protective in sepsis.     

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.     

Studies of molecular mechanism of tenofovir against 3TC- and AZT-resistance mutant HIV-1 reverse transcriptase

Molecular modeling study shows that conformational flexibility of acyclic nature of TFV provides energetically indistinguishable multiple conformations, which do not experience the cross-resistance conferred by mutant RTs. TFV-DP is located far away from the bulky side chain of Val184 in M184V RT and tenofovir is readily translocated without steric hindrance with Asp185 after incorporation into the growing primer chain complexed with AZT-resistant RT.     

ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.     

Molecular dissection of QTL governing grain size traits employing association and linkage mapping in Basmati rice

Grain size is one of the key traits that determines the quality of Basmati rice from the consumers’ as well as the traders’ point of view. Though many genes governing grain size have been identified in indica and japonica, little work has been done in Basmati rice. The present study aims at dissection of a QTL region governing grain size traits in Basmati employing association and linkage mapping approaches. Association mapping revealed that three markers, i.e., RM 6024 (grain breadth), RM1237 and RM18582 (grain length-breadth ratio), which cover 889 kb in the targeted QTL region have been significantly associated with grain size traits. Using linkage mapping, the targeted QTL region has been further delimited to a physical distance of 268 kb that comprises 24 annotated genes. The gene expression analysis of parents, revealed 19 genes differentially expressing within the QTL. Of them, 15 genes showed high expression in Basmati370, while four were expressed in Jaya, and whereas five genes did not show any differential expression between parents. Among differentially expressed genes, a highly expressed gene in Basmati370, Os05g0374200 (Cytokinin dehydrogenase 1 precursor) seems to be involved in accumulation of cytokinins, thus affecting the grain size. Therefore, our findings demonstrated that by complimenting association and linkage mapping, it is likely to dissect a QTL governing grain size traits in Basmati rice and also the QTL could be a potential target for marker-assisted breeding and map-based cloning studies.