Starch with high amylose and low in vitro digestibility increases short-chain fatty acid absorption, reduces peak insulin secretion, and modulates incretin secretion in pigs

Diets containing different starch types affect peripheral glucose and insulin responses. However, the role of starch chemistry in kinetics of nutrient absorption and insulin and incretin secretion is poorly understood. Four portal vein-catheterized pigs (35.0 ± 0.2 kg body weight) consumed 4 diets containing 70% purified starch [0-63.2% amylose content and 0.22 (slowly) to 1.06%/min (rapidly) maximum rate of in vitro digestion] for 7-d periods in a 4 × 4 Latin square. On d 7, blood was collected for 12 h postprandial with simultaneous blood flow measurement for determining the net portal appearance (NPA) of nutrients and hormones. The NPA of glucose, insulin, C-peptide, and glucose-dependent insulinotropic polypeptide (GIP) during 0-4 h postprandial were lower (P < 0.05) and those of butyrate and total SCFA were higher (P < 0.05) when pigs consumed the diet containing slowly digestible compared with rapidly digestible starch. The peak NPA of insulin occurred prior to that of glucose when pigs consumed diets containing rapidly digestible starch. The kinetics of insulin secretion had a linear positive relation with kinetics of NPA of glucose (R(2) = 0.50; P < 0.01). In conclusion, starch with high amylose and low in vitro digestibility decreases the kinetics of glucose absorption and insulin and GIP secretion and increases SCFA absorption and glucagon-like peptide-1 secretion. In conclusion, starch with high amylose content and a lower rate and extent of in vitro digestion decreased glucose absorption and insulin secretion and increased SCFA absorption.     

Clinical outcomes in adult patients with aplastic anemia: A single institution experience

Newer treatment modalities are being investigated to improve upon historical outcomes with standard immunosuppressive therapy (IST) in aplastic anemia (AA). We analyzed outcomes of adult patients with AA treated with various combinatorial anti‐thymoglobulin‐based IST regimens in frontline and relapsed/refractory (R/R) settings. Pretreatment and on‐treatment clinical characteristics were analyzed for relationships to response and outcome. Among 126 patients reviewed, 95 were treatment‐naïve (TN) and 63, R/R (including 32 from the TN cohort); median ages were 49 and 50 years, respectively. Overall survival (OS) was superior in IST responders (P < .001). Partial response to IST was associated with shorter relapse‐free survival (RFS), as compared with complete response (P = .03). By multivariate analysis, baseline platelet and lymphocyte count predicted for IST response at 3 and 6 months, respectively. While additional growth factor interventions led to faster count recovery, there were no statistically significant differences in RFS or OS across the various frontline IST regimens (i.e., with/without G‐CSF or eltrombopag). While marrow cellularity did not correlate with peripheral‐blood counts at 3 months, cytomorphological assessment revealed dyspoietic changes in all nonresponders with hypercellular‐marrow indices. Covert dysplasia, identified through early bone marrow assessment, has implications on future therapy choices after IST failure. Salvage IST response depended upon prior response to ATG: prior responders (46%) vs. primary refractory (0%) (P < .01). In the R/R setting, there was no survival difference between IST and allogeneic stem cell transplant groups, with a trend toward superior OS in the former. Transplant benefits in the R/R setting may be underrealized due to transplant‐related mortality.     

Ras is an indispensable coregulator of the class IB phosphoinositide 3-kinase p87/p110γ

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kγ consists of a catalytic p110γ and a regulatory p87^PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein βγ (Gβγ)-mediated PI3Kγ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110γ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kγ-complemented bone marrow–derived mast cells (BMMCs) from p110γ^-/- mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein–coupled receptor (GPCR)-induced PI3Kγ activation. Although p101 strongly interacted with Gβγ, thereby mediating PI3Kγ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gβγ-dependent membrane recruitment of p87/p110γ by direct interaction with p110γ, suggesting the indispensability of Ras for activation of p87/p110γ. Consequently, interference with Ras signaling indeed selectively blocked p87/p110γ, but not p101/p110γ, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110γ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kγ that could open up new possibilities for therapeutic intervention.     

DermatoFibroSarcoma Protuberans: A case report of a complete cure

DermatoFibroSarcoma Protuberans (DFSP) is a rare recurrent fibrohistiocytic tumor. Given the limitation of available diagnostic modalities in a resource poor setting, diagnosis can be confusing. As most of the tumors recur with time, our case of complete cure was interesting phenomenon observed in our case.     

Computational identification of natural product inhibitors against EGFR double mutant (T790M/L858R) by integrating ADMET,machine learning,molecular docking and a dynamics approach

Double mutated epidermal growth factor receptor is a clinically important target for addressing drug resistance in lung cancer treatment. Therefore, discovering new inhibitors against the T790M/L858R (TMLR) resistant mutation is ongoing globally. In the present study, nearly 150 000 molecules from various natural product libraries were screened by employing different ligand and structure-based techniques. Initially, the library was filtered to identify drug-like molecules, which were subjected to a machine learning based classification model to identify molecules with a higher probability of having anti-cancer activity. Simultaneously, rules for constrained docking were derived from three-dimensional protein–ligand complexes and thereafter, constrained docking was undertaken, followed by HYDE binding affinity assessment. As a result, three molecules that resemble interactions similar to the co-crystallized complex were selected and subjected to 100 ns molecular dynamics simulation for stability analysis. The interaction analysis for the 100 ns simulation period showed that the leads exhibit the conserved hydrogen bond interaction with Gln791 and Met793 as in the co-crystal ligand. Also, the study indicated that Y-shaped molecules are preferred in the binding pocket as it enables them to occupy both pockets. The MMGBSA binding energy calculations revealed that the molecules have comparable binding energy to the native ligand. The present study has enabled the identification of a few ADMET adherent leads from natural products that exhibit the potential to inhibit the double mutated drug-resistant EGFR.

Identification of ADMET adherent natural products that have high affinity for double mutated drug-resistant EGFR.     

Chronic ethanol induces inhibition of antigen-specific CD8+ but not CD4+ immunodominant T cell responses following Listeria monocytogenes inoculation

Chronic ethanol consumption results in immunodeficiency. Previous work with chronic ethanol-fed mice has shown reduced splenic weight and cellularity, including reduced numbers of CD8+ T cells. However, antigen-specific CD8+ and CD4+ T cell responses in chronic ethanol-fed mice have been studied relatively little. We have used an attenuated Listeria monocytogenes strain DPL 1942 (LM DeltaactA) to inoculate mice and subsequently used CD4+ and CD8+ immunodominant peptides of LM to measure the CD4+ and CD8+ T cell responses after chronic ethanol exposure. We found no major differences between control and ethanol-fed mice in the kinetics and persistence of antigen-specific CD4+ T cells in response to an immunodominant LM peptide, as measured by intracellular IFN-gamma staining. In contrast to CD4+ responses, three methods of in vitro antigen presentation indicated that the primary response of CD8+ T cells to several different epitopes was reduced significantly in mice chronically fed ethanol. Antigen-specific CD8+ T cells were also reduced in chronic ethanol-fed mice during the contraction phase of the primary response, and memory cells evaluated at 29 and 60 days after inoculation were reduced significantly. BrdU proliferation assays showed that in vivo proliferation of CD8+ T cells was reduced in ethanol-fed mice, and IL-2-dependent in vitro proliferation of naive CD8+ T cells was also reduced. In conclusion, these results suggest that antigen-specific CD4+ T cell responses to LM are affected little by chronic ethanol consumption; however, antigen-specific CD8+ T cell responses are reduced significantly, as are in vivo and in vitro proliferation. The reduction of antigen-specific CD8+ T cells may contribute strongly to the immunodeficiency caused by ethanol abuse.     

Novel Roles for Caspase-8 in IL-1β and Inflammasome Regulation

Caspase-8 is an initiator and apical activator caspase that plays a central role in apoptosis. Caspase-8–deficient mice are embryonic lethal, which makes study of caspase-8 in primary immune cells difficult. Recent advances have rescued caspase-8–deficient mice by crossing them to mice deficient in receptor-interacting serine-threonine kinase 3 (RIPK3). These genetic tools have made it possible to study the role of caspase-8 in vivo and in primary immune cells. Several recent studies have identified novel roles for caspase-8 in modulating IL-1β and inflammation, showing that caspase-8 directly regulates IL-1β independent of inflammasomes or indirectly through the regulation of inflammasomes, depending on the stimulus or stimuli that initiate the signaling cascade. Here, we address recent findings on caspase-8 and its role in modulating IL-1β and inflammation.Caspase-8 is an initiator and apical activator caspase that plays a central role in apoptosis. It consists of two N-terminal death effector domains (DEDs), which are followed by a large (p18) and a small (p10) protease subunit at the C-terminal end (Figure 1). First described in 1996, caspase-8 is essential for death receptor–induced activation of the extrinsic cell-death pathway.^1,2 On activation of death receptors (CD95, TNFR1, or DR5), caspase-8 is recruited to the receptors via the adaptor protein FAS-associated death domain (FADD). Caspase-8 and FADD both contain DEDs, which mediate DED–DED homotypic interactions and coordinate complex formation of death receptors. Caspase-8 homodimer formation in this complex results in activation and autocleavage, which further stabilizes the active dimer. Active caspase-8 then processes and cleaves downstream executioner caspases, or the BCL2 family member BID, to initiate apoptosis. Because apoptosis is central for development and survival of the host, caspase-8 activation is tightly regulated. cFLIP, a homolog of caspase-8, blocks caspase-8 apoptotic function by forming heterodimeric complexes³ (Figure 1). It has also been proposed that caspase-8 is cleaved, and in some instances activated by other caspases, such as caspase-3^4,5 and caspase-6,^5,6 as well as by the proteases granzyme B⁷ and cathepsin D.⁸Open in a separate windowFigure 1Role for caspase-8 (CASP8) in inducing apoptosis and regulating signaling pathways. A: Procaspase-8 consists of two N-terminal death-effector domain (DED) prodomains, which are followed by the catalytic subunits p18 and p10, respectively. On dimerization, caspase-8 is cleaved at the sites between the DED and p18, and between p18 and p10. B: Death receptor (CD95, TNFR1, DR5) engagement with the respective ligand [CD95L, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis inducing ligand (TRAIL)] results in recruitment of FAS-associated death domain (FADD) and caspase-8 homodimers. Activation of caspase-8 results in induction of apoptosis. cFLIP can bind to caspase-8 to form cFLIP–caspase-8 heterodimers. The formation of cFLIP–caspase-8 heterodimers inhibits apoptosis. C: Activation of Toll-like receptor 4 (TLR4) or TLR2 results in recruitment of TIR domain-containing adaptor-inducing interferon-β (TRIF) and myeloid differentiation primary response protein MyD88 (MyD88) to the receptors. TRIF and MyD88 signaling results in downstream nuclear factor-κB (NF-κB) signaling events that induce mRNA expression of pro–IL-1β and NLRP3. Evidence suggests that FADD and caspase-8 are required for optimal expression of pro–IL-1β and NLRP3 mRNA, possibly through their role in NF-κB activation. LPS, lipopolysaccharide; PGN, peptidoglycan.The importance of caspase-8 is highlighted by the fact that knockout mice die at approximately embryonic day 10.5.⁹ In seminal studies, the Mocarski¹⁰ and Green¹¹ research groups showed that deletion of receptor-interacting serine-threonine kinase 3 (RIPK3, involved in necroptotic cell death) rescues caspase-8 deficient mice. These studies established a nonapoptotic role for caspase-8, namely, to rescue the lethality induced by RIPK3-mediated pathways. The generation of double-knockout Ripk3^−/−Casp8^−/− mice has provided an invaluable tool for investigating the role of caspase-8 in vivo and in primary immune cells.Here, we discuss inflammasome-mediated IL-1β production and the novel roles of caspase-8 in modulating inflammasomes, IL-1β, and inflammation.