Challenges With the Use of Digital Sham: Systematic Review and Recommendations.

BACKGROUND: Digital therapeutics (DTx) are software-based products that prevent, manage, or treat a medical condition and are delivered through a smartphone app, web application, or wearable device. Clinical trials assessing DTx pose challenges, foremost among which is designing appropriate digital shams (or digital placebos), which should ideally mimic DTx (in terms of design, components, and duration of treatment) while omitting the active principle or component. OBJECTIVE: The objective of our review was to understand how digital shams are being used in clinical research on DTx in neuroscience, which is the most common therapy area for DTx. METHODS: We conducted a systematic literature review of DTx in neuroscience (including neurodevelopmental, neurodegenerative, and psychiatric disorders) with a focus on controlled clinical trials involving digital shams. Studies were identified from trial registries (ClinicalTrials.gov, the European Union Clinical Trials Register, and Trial Trove) and through structured searches in MEDLINE and Embase (both via the Embase website) and were limited to articles in English published from 2010 onward. These were supplemented by keyword-based searches in PubMed, Google, and Google Scholar and bibliographic searches. Studies assessing DTx in neuroscience (including neurodevelopmental, neurodegenerative, and psychiatric disorders) were included. Details related to the publication, DTx, comparator, patient population, and outcomes were extracted and analyzed. RESULTS: Our search criteria identified 461 neuroscience studies involving 213 unique DTx. Most DTx were extended reality based (86/213, 40.4%) or mobile device based (56/213, 26.3%); 313 were comparative, of which 68 (21.7%) used shams. The most common therapeutic areas assessed in these studies were stroke (42/213, 19.7%), depression (32/213, 15%), and anxiety (24/213, 11.3%). The most common treatments were cognitive behavioral therapy or behavioral therapy (67/213, 32.4%), physical rehabilitation (60/213, 28.2%), and cognitive training (41/213, 19.2%). We identified the following important issues related to the use of digital shams in neuroscience: shams were not validated before use in studies, they varied widely in design (from being nearly identical to the DTx to using different software programs altogether), and the level of patient engagement or satisfaction with the sham and the impact of the sham on study outcomes were infrequently reported. CONCLUSIONS: Digital shams are critical for the clinical development of DTx in neuroscience. Given the importance of sham controls in evaluating DTx efficacy, we provide recommendations on the key information that should be reported in a well-designed DTx trial and propose an algorithm to allow the correct interpretation of DTx study results. Sham-controlled studies should be routinely used in DTx trials-in early-phase studies-to help identify DTx active components and-in late-phase studies-to confirm the efficacy of DTx. The use of shams early in development will ensure that the appropriate sham control is used in later confirmatory trials.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells.

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 µm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 µm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 µm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 µm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.

Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells.

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1ß, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

TCF 4 tumor suppressor: a molecular target in the prognosis of sporadic colorectal cancer in humans.

BACKGROUND: A huge array of function is played by the Wnt/ß-catenin signaling pathway in development by balancing gene expression through the modulation of cell-specific DNA binding downstream effectors such as T-cell factor/lymphoid enhancer factor (TCF/LEF). The ß-catenin/TCF-4 complex is a central regulatory switch for differentiation and proliferation of intestinal cells (both normal and malignant). Thus, in the present study we evaluated each of 60 cases of sporadic adenocarcinoma, alongside adjoining and normal mucosa specimens of colorectum in humans, for mutation and expression analysis of the gene coding for TCF-4 protein. METHODS: DNA sequencing following PCR amplification and SSCP analysis (single strand conformation polymorphism) was employed to detect TCF-4 gene mutations in the case of exon 1. Quantitative real-time (qRT) PCR, immunohistochemistry (IHC), confocal microscopy and western blot analysis were used to detect TCF-4 gene/protein expression. RESULTS: Sequencing analysis confirmed 5/60 patients with a point mutation in exon 1 of the TCF-4 gene in tumor samples. mRNA expression using qRT-PCR showed approximately 83% decreased TCF-4 mRNA expression in tumor tissue and adjoining mucosa compared to normal mucosa. Similarly, a significant decrease in protein expression using IHC showed decreased TCF-4 protein expression in tumor tissue and adjoining mucosa compared to normal mucosa, which also corresponds to some important clinicopathological factors, including disease metastasis and tumor grade. Mutational alterations and downregulation of TCF-4 mRNA and hence decreased expression of TCF-4 protein in tumors suggest its involvement in the pathogenesis of CRC. CONCLUSIONS: A remarkable decrease in TCF-4 mRNA and protein expression was detected in tumorous and adjoining tissues compared to normal mucosa. Hence the alterations in genomic architecture along with downregulation of TCF-4 mRNA and decreased expression of TCF-4 protein in tumors, which is in accordance with clinical features, suggest its involvement in the pathogenesis of CRC. Thus, deregulation and collaboration of TCF-4 with CRC could be a concrete and distinctive feature in the prognosis of the disease at an early stage of development.

Mechanisms of Niemann-Pick type C1 Like 1 protein degradation in intestinal epithelial cells.

Intestinal Niemann-Pick C1 Like 1 (NPC1L1) protein plays a key role in cholesterol absorption. A decrease in NPC1L1 expression has been implicated in lowering plasma cholesterol and mitigating the risk for coronary heart disease. Little is known about the mechanisms responsible for NPC1L1 protein degradation that upon activation may lead to a reduction in NPC1L1 protein levels in intestinal epithelial cells (IECs). In current studies, the human intestinal Caco-2 and HuTu-80 cell lines expressing NPC1L1-hemagglutinin fusion protein were used to investigate the mechanisms of NPC1L1 protein degradation. Incubation with the proteasome inhibitors MG-132 and lactacystin (10 µM, 24 h) significantly increased NPC1L1 protein levels in IECs. Also, the inhibition of the lysosomal pathway with bafilomycin A1 (80 nM, 24 h) resulted in a significant increase in NPC1L1 protein levels. Immunoprecipitation studies showed that NPC1L1 protein is both a poly- and monoubiquinated polypeptide and that the inhibition of the proteasomal pathway remarkably increased the level of the polyubiquinated NPC1L1. The surface expression of NPC1L1 was increased by the inhibition of both proteasomal and lysosomal pathways. Furthermore, the pharmacological inhibition of mitogen-activated protein kinase pathway (PD-98059, 15 µM, 24 h) and siRNA silencing of ERK1/2 resulted in a significant decrease in NPC1L1 protein levels in IECs. In conclusion, our results showed that basal level of intestinal cholesterol transporter NPC1L1 protein is modulated by both ubiquitin proteasome- and lysosome-dependent degradation as well as by ERK1/2-dependent pathway. The modulation of these pathways may provide novel clues for therapeutic intervention to inhibit cholesterol absorption and lower plasma cholesterol.

Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression.

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 µM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Overactivation of intestinal sterol response element-binding protein 2 promotes diet-induced nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH, suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the sterol response element-binding protein-2 (SREBP-2) transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP-2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine-specific overexpression of active SREBP-2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and their wild-type littermates were fed a regular chow diet or a high-fat, high-cholesterol (HFHC) diet (15% fat, 1% cholesterol) for 15 wk. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice compared with wild-type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice compared with wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver.NEW & NOTEWORTHY The current study highlights the role of overactivation of intestinal SREBP-2 transcription factor in the progression of hepatic fibrosis associated with diet-induced NASH. Mice with intestine-specific overexpression of SREBP-2 demonstrated more inflammation and severe fibrosis in the liver in response to 15 wk of being fed a high-cholesterol, high-fat diet as compared with their wild-type littermates. These data demonstrate a novel link between intestinal regulatory processes of cholesterol metabolism and the pathogenesis of fatty liver diseases.

Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions.

A major goal of protein folding studies is to understand the structural basis of the coupling between stabilizing interactions, which leads to cooperative conformational change. The goal is challenging because of the difficulty in simultaneously measuring global cooperativity by determining population distributions of the conformations present, and the structures of these conformations. Here, hydrogen exchange (HX) into the small protein monellin was carried out under conditions where structure-opening is rate limiting for most backbone amide sites. Detection by mass spectrometry allowed characterization of not only segment-specific structure-opening rates but also the cooperativity of unfolding of the different secondary structural segments of the protein. The segment-specific pattern of HX reveals that the backbone hydrogen-bonding network disassembles in a structurally diffuse, asynchronous manner. A comparison of the site-specific transient opening rates of secondary and tertiary structure in the protein provides a structural rationale for the observation that unfolding is hierarchical and describable by exponential kinetics, despite being diffuse. Since unfolding was studied in native conditions, the sequence of events during folding in the same conditions will be the reverse of the sequence of events observed during unfolding. Hence, the formation of secondary structural units during folding would also occur in a non-cooperative, diffuse, and asynchronous manner.

N-glycosylation is essential for ileal ASBT function and protection against proteases.

The bile acid transporter ASBT is a glycoprotein responsible for active absorption of bile acids. Inhibiting ASBT function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. Deglycosylation of ASBT was shown to decrease its function. However, the exact roles of N-glycosylation of ASBT, and how it affects its function, is not known. Current studies investigated the roles of N-glycosylation in ASBT protein stability and protection against proteases utilizing HEK-293 cells stably transfected with ASBT-V5 fusion protein. ASBT-V5 protein was detected as two bands with molecular mass of ~41 and ~35 kDa. Inhibition of glycosylation by tunicamycin significantly decreased ASBT activity and shifted ASBT bands to ~30 kDa, representing a deglycosylated protein. Treatment of total cellular lysates with PNGase F or Endo H glycosidases showed that the upper 41-kDa band represents a fully mature N-acetylglucosamine-rich glycoprotein and the lower 35-kDa band represents a mannose-rich core glycoprotein. Studies with the glycosylation deficient ASBT mutant (N10Q) showed that the N-glycosylation is not essential for ASBT targeting to plasma membrane. However, mature glycosylation significantly increased the half-life and protected ASBT protein from digestion with trypsin. Incubating the cells with high glucose (25 mM) for 48 h increased mature glycosylated ASBT along with an increase in its function. These results unravel novel roles for N-glycosylation of ASBT and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in ASBT function in diabetes mellitus.

Tuning Cooperativity on the Free Energy Landscape of Protein Folding.

Understanding the origin of the cooperativity seemingly inherent in a folding or unfolding reaction has been a major challenge. In particular, the relationship between folding cooperativity and stability is poorly understood. In this study, native state hydrogen exchange in conjunction with mass spectrometry has been used to explore the free energy landscape accessible to the small protein monellin, when the stability of the protein is varied. Mass distributions obtained in the EX1 limit of exchange have allowed a direct distinction between correlated (cooperative) and uncorrelated (noncooperative) structure-opening processes. Under conditions where the native protein is maximally stable, a continuum of partially unfolded states is gradually sampled before the globally unfolded state is transiently sampled. Under conditions that stabilize the unfolded state of the protein, the slowest structure-opening reactions leading to complete unfolding become cooperative. The present study provides experimental evidence for a gradual uphill unfolding transition on a very slow time scale, in the presence of a large free energy difference between the native and unfolded states. The results suggest that the cooperativity that manifests itself in protein folding and unfolding reactions carried out in the presence of denaturant might merely be a consequence of the effect of the denaturant on the unfolded state and transition state stabilities.

Epigenetic modulation of intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) gene expression by DNA methylation.

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.

High-energy intermediates in protein unfolding characterized by thiol labeling under nativelike conditions.

A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.

Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India.

To determine the etiological factors of human colorectal cancer (CRC) we assessed the frequency and prognostic significance of hMLH1 and hMSH2 genes in conjunction with hMLH1 and hMSH2 protein expression in 30 Indian CRC patients. The protein expression and promoter methylation of hMLH1 and hMSH2; Mismatch Repair genes (MMR) were analyzed by immunohistochemistry and methylation-specific PCR (MSP), respectively. A loss of hMLH1 expression was recognized in 4(13.3%) and loss of hMSH2 expression was recognized in 2(6.6%) of 30 CRC cases whereas 50% tumors showed reduced expression of hMLH1 and 33.3% showed reduced expression of hMSH2 protein. One tumor showed a loss of both hMLH1 and hMSH2 expression. Normal nuclear staining pattern of hMLH1 and hMSH2 was observed in almost all the adjoining and normal mucosa. Promoter hypermethylation of the hMLH1 gene was detected in 15 of 30 CRC cases (50%) and of hMSH2 gene was only in 3 of 30 CRC cases (10%). No promoter methylation of hMLH1 and hMSH2 genes was observed in adjoining and normal mucosa. Combination of methylation of hMLH1 and hMSH2 gene was observed in two tumors (6.6%). A significant correlation between histological grade of the tumor, methylation and expression of hMLH1 gene (p < 0.05) was observed. Normal expression of hMLH1 and hMSH2 was seen in all of the unmethylated tumors (100%). Nuclear staining and promoter methylation of hMLH1 and hMSH2 did not significantly influence survival. hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression. In contrast, hMSH2 methylation was infrequent. These findings suggest that the inactivation of MMR gene expression probably via hypermethylation may lead to inactivation of their functions which finally leads to tumor aggressiveness and the immunostaining of hMLH1 protein can be used as a prognostic factor for determining the grade of the tumor.

Effects of Soil Rhizobia Abundance on Interactions between a Vector, Pathogen, and Legume Plant Host.

Soil rhizobia promote nitrogen fixation in legume hosts, maximizing their tolerance to different biotic stressors, plant biomass, crop growth, and yield. While the presence of soil rhizobia is considered beneficial for plants, few studies have assessed whether variation in rhizobia abundance affects the tolerance of legumes to stressors. To address this, we assessed the effects of variable soil rhizobia inoculum concentrations on interactions between a legume host (Pisum sativum), a vector insect (Acyrthosiphon pisum), and a virus (Pea enation mosaic virus, PEMV). We showed that increased rhizobia abundance reduces the inhibitory effects of PEMV on the nodule formation and root growth in 2-week-old plants. However, these trends were reversed in 4-week-old plants. Rhizobia abundance did not affect shoot growth or virus prevalence in 2- or 4-week-old plants. Our results show that rhizobia abundance may indirectly affect legume tolerance to a virus, but effects varied based on plant age. To assess the mechanisms that mediated interactions between rhizobia, plants, aphids, and PEMV, we measured the relative expression of gene transcripts related to plant defense signaling. Rhizobia concentrations did not strongly affect the expression of defense genes associated with phytohormone signaling. Our study shows that an abundance of soil rhizobia may impact a plant's ability to tolerate stressors such as vector-borne pathogens, as well as aid in developing sustainable pest and pathogen management systems for legume crops. More broadly, understanding how variable rhizobia concentrations can optimize legume-rhizobia symbiosis may enhance the productivity of legume crops.

Regulation of intestinal serotonin transporter expression via epigenetic mechanisms: role of HDAC2.

The serotonin (5-HT) transporter (SERT) facilitates clearance of extracellular 5-HT by its uptake and internalization. Decreased expression of SERT and consequent high 5-HT levels have been implicated in various diarrheal disorders. Thus, appropriate regulation of SERT is critical for maintenance of 5-HT homeostasis in health and disease. Previous studies demonstrated that SERT is regulated via posttranslational and transcriptional mechanisms. However, the role of epigenetic mechanisms in SERT regulation is not known. Current studies investigated the effects of histone deacetylase (HDAC) inhibition on SERT expression and delineated the mechanisms. Treatment of Caco-2 cells with the pan-HDAC inhibitors butyrate (5 mM) and trichostatin (TSA, 1 µM) decreased SERT mRNA and protein levels. Butyrate- or TSA-induced decrease in SERT was associated with decreased activity of human SERT (hSERT) promoter 1 (upstream of exon 1a), but not hSERT promoter 2 (upstream of exon 2). Butyrate + TSA did not show an additive effect on SERT expression, indicating that mechanisms involving histone hyperacetylation may be involved. Chromatin immunoprecipitation assays demonstrated enrichment of the hSERT promoter 1 (flanking nt -250/+2) with tetra-acetylated histone H3 or H4, which was increased (~3-fold) by butyrate. Interestingly, specific inhibition of HDAC2 (but not HDAC1) utilizing small interfering RNA decreased SERT mRNA and protein levels. The decrease in SERT expression by HDAC inhibition was recapitulated in an in vivo model. SERT mRNA levels were decreased in the ileum and colon of mice fed pectin (increased availability of butyrate) compared with controls fed a fiber-free diet (~50-60%). Our results identify a novel role of HDAC2 as a regulator of SERT gene expression in intestinal epithelial cells.

D-Glucose modulates intestinal Niemann-Pick C1-like 1 (NPC1L1) gene expression via transcriptional regulation.

The expression of intestinal Niemann-Pick C1-like 1 (NPC1L1) cholesterol transporter has been shown to be elevated in patients with diseases associated with hypercholesterolemia such as diabetes mellitus. High levels of glucose were shown to directly increase the expression of NPC1L1 in intestinal epithelial cells, but the underlying mechanisms are not fully defined. The present studies were, therefore, undertaken to examine the transcriptional regulation of NPC1L1 expression in human intestinal Caco2 cells in response to glucose. Removal of glucose from the culture medium of Caco2 cells for 24 h significantly decreased the NPC1L1 mRNA, protein expression, as well as the promoter activity. Glucose replenishment significantly increased the promoter activity of NPC1L1 in a dose-dependent manner compared with control cells. Exposure of Caco2 cells to nonmetabolizable form of glucose, 3-O-methyl-d-glucopyranose (OMG) had no effect on NPC1L1 promoter activity, indicating that the observed effects are dependent on glucose metabolism. Furthermore, glucose-mediated increase in promoter activity was abrogated in the presence of okadaic acid, suggesting the involvement of protein phosphatases. Glucose effects on several deletion constructs of NPC1L1 promoter demonstrated that cis elements mediating the effects of glucose are located in the region between -291 and +56 of NPC1L1 promoter. Consistent with the effects of glucose removal on NPC1L1 expression in Caco2 cells, 24-h fasting resulted in a significant decrease in the relative expression of NPC1L1 in mouse jejunum. In conclusion, glucose appears to directly modulate NPC1L1 expression via transcriptional mechanisms and the involvement of phosphatase-dependent pathways.

Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.

The Intricate Role of Ecdysis Triggering Hormone Signaling in Insect Development and Reproductive Regulation.

Insect growth is interrupted by molts, during which the insect develops a new exoskeleton. The exoskeleton confers protection and undergoes shedding between each developmental stage through an evolutionarily conserved and ordered sequence of behaviors, collectively referred to as ecdysis. Ecdysis is triggered by Ecdysis triggering hormone (ETH) synthesized and secreted from peripheral Inka cells on the tracheal surface and plays a vital role in the orchestration of ecdysis in insects and possibly in other arthropod species. ETH synthesized by Inka cells then binds to ETH receptor (ETHR) present on the peptidergic neurons in the central nervous system (CNS) to facilitate synthesis of various other neuropeptides involved in ecdysis. The mechanism of ETH function on ecdysis has been well investigated in holometabolous insects such as moths Manduca sexta and Bombyx mori, fruit fly Drosophila melanogaster, the yellow fever mosquito Aedes aegypti and beetle Tribolium castaneum etc. In contrast, very little information is available about the role of ETH in sequential and gradual growth and developmental changes associated with ecdysis in hemimetabolous insects. Recent studies have identified ETH precursors and characterized functional and biochemical features of ETH and ETHR in a hemimetabolous insect, desert locust, Schistocerca gregaria. Recently, the role of ETH in Juvenile hormone (JH) mediated courtship short-term memory (STM) retention and long-term courtship memory regulation and retention have also been investigated in adult male Drosophila. Our review provides a novel synthesis of ETH signaling cascades and responses in various insects triggering diverse functions in adults and juvenile insects including their development and reproductive regulation and might allow researchers to develop sustainable pest management strategies by identifying novel compounds and targets.