Role of cryptochromes from the marine worm in the circadian/circalunar clock

In most organisms including humans, physiology and behavior are coordinated by an internal molecular clock. These rhythms are synchronized with the 24h solar day and termed as circadian rhythms. At a molecular level, the circadian clock is governed by the interplay between several proteins such as CLOCK, CYCLE, PERIOD and CRY and is regulated by transcription-translation feedback loop. These regulatory proteins are partially conserved between several insect and mammalian systems indicating a possible evolutionary function. Many organisms align their behavior not only to the changes of the sun but also to the cycles of the moon (monthly timing). The bristle worm Platynereis was among the first organisms for which the entrainment to a lunar cycle was shown to only depend on nocturnal light stimuli. Thus, Platynereis dumerilli has a circalunar (monthly) clock in addition to the circadian (daily) clock. The locomotor activity of the worm is under the control of the circadian clock. The circalunar clock regulates the maturation of Platynereis adjusting its reproduction to the lunar phase. Platynereis is a suitable model to study the interplay between circadian and circalunar clock components and the influence of this on the behavior of the organism. It has been established that the circalunar clock of the worm affects the circadian clock at several levels and the circalunar clock persists even when the circadian clock is disrupted. Cryptochromes are well studied components of the circadian clock which have the potential to function as light receptors in some species. This study focuses on the characterization of the cryptochromes from Platynereis dumerilli to provide insights to their role in circadian and/or circalunar clocks. Platynereis dumerilli possess 3 different kinds of cryptochromes, one each similar to the drosophila (L-CRY), mammalian repressor (TR-CRY) and plant (P-CRY) cryptochromes. Studies with S2 cells also established that TR-CRY functions as a transcriptional repressor cryptochrome as in mammals. However, the role of L-CRY and P-CRY remain to be elucidated. In this thesis, characterization of L-CRY and P-CRY has been carried out with purified proteins. Here, we have purified the Platynereis L-CRY and P-CRY proteins and analyzed them by UV/VIS- and fluorescence spectroscopy, high performance liquid chromatography (HPLC) and static light scattering (MALS/SLS) to determine the oligomeric state, their cofactors bound and to propose their photoreaction mechanisms. Our analyses suggest possible roles of Platynereis L-CRY and P-CRY as flavin-dependent photoreceptors. SAXS and electron microscopy analysis provided structural information regarding L-CRY with the detection of a novel dimer interface. To gain further insights into the functional role of these cryptochromes, a mass spectrometry-based approach helped to determine potential interacting partners. The next level of understanding will be to discern how these different cryptochromes and their interactors are molecularly connected and to what extent underlying molecular clocks share same components.197 page

Enhancing TCP Performance In Wired-Cum-Wireless Networks

Increasing popularity for mobile devices has prompted industrial and academic research towards improving the performance of wireless applications. Transmission Control Protocol (TCP) plays an important role in defining a network’s performance, and its use in wireless networks has exposed several inadequacies in its operation. Tight coupling of TCP’s error and congestion control mechanisms has proven to be incompatible with the unique characteristics of wireless channels. TCP, designed for wired networks, assumes any loss of packet to be an indication of congestion in the network. Wireless networks exhibit a higher bit error rate, low and varying bandwidth, and disconnections of hosts due to mobility. All of the aforementioned reasons can result in random packet loss which is misinterpreted as a sign of congestion by TCP. Such erroneous triggering of congestion control measures can unnecessarily reduce TCP throughput. In this report, we will delve deeper into TCP’s operation, and discuss its performance issues in wired-cum-wireless networks. We also present a survey of existing schemes that tackle these issues, and introduce a new scheme called TCP-ECN to enhance TCP performance in wireless networks. The essence of the new scheme is to use Explicit Congestion Notification to enable the wireless host to distinguish between wired and wireless losses. Another facet of our scheme is to allow the base station to “freeze” the sender when it notices an imminent disconnection of the mobile host. The objective of TCP-ECN is to insulate the TCP sender from the idiosyncrasies of the wireless channel. We have both simulated and implemented the new scheme. This report details the new scheme in depth, and analyzes the test results obtained

Osteocalcin Is Independently Associated with C-Reactive Protein during Lifestyle-Induced Weight Loss in Metabolic Syndrome

Bone-derived osteocalcin has been suggested to be a metabolic regulator. To scrutinize the relation between osteocalcin and peripheral insulin sensitivity, we analyzed changes in serum osteocalcin relative to changes in insulin sensitivity, low-grade inflammation, and bone mineral density following lifestyle-induced weight loss in individuals with metabolic syndrome (MetS). Participants with MetS were randomized to a weight loss program or to a control group. Before and after the 6-month intervention period, clinical and laboratory parameters and serum osteocalcin levels were determined. Changes in body composition were analyzed by dual-energy X-ray absorptiometry (DXA). In participants of the intervention group, weight loss resulted in improved insulin sensitivity and amelioration of inflammation. Increased serum levels of osteocalcin correlated inversely with BMI (r = −0.63; p < 0.001), total fat mass (r = −0.58, p < 0.001), total lean mass (r = −0.45, p < 0.001), C-reactive protein (CRP) (r = −0.37; p < 0.01), insulin (r = −0.4; p < 0.001), leptin (r = −0.53; p < 0.001), triglycerides (r = −0.42; p < 0.001), and alanine aminotransferase (ALAT) (r = −0.52; p < 0.001). Regression analysis revealed that osteocalcin was independently associated with changes in CRP but not with changes in insulin concentration, fat mass, or bone mineral density, suggesting that weight loss-induced higher serum osteocalcin is primarily associated with reduced inflammation

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen

Role of overminus therapy in intermittent exotropia

Aim: The aim is to study the change in Newcastle Control Score (NCS) and angle of deviation after a trial of overminus therapy in intermittent exotropia (IXT). Context: Overminus therapy in IXT. Settings and Designs: This prospective observational study was conducted at Comtrust Eye Hospital, Calicut, Kerala, which is a tertiary eye care center from July 2015 to May 2017. Materials and Methods: Fifty-three children with IXT, aged 1–5 years, were recruited. NCS scores and angle of deviation were recorded. Treatment was instigated with the minimum minus lens required to achieve control of the manifest deviation. NCS and angle of deviation were recorded with the overminus glasses at 6 weeks, 6 months, and 1 year. Statistical Analysis: Data were tabulated and analyzed using the SPSS software (SPSS statistics for Windows 17.0 Chicago, SPSS Inc). Paired t-test was used to compare change in NCS and angle of deviation before and after treatment. P < 0.05 was taken as statistically significant. Results: Fifty-three children were recruited, 31 females and 22 males with a mean age of 3.6 ± 1.633 years. Majority were of the basic type of IXT. The NCS at 1 year showed significant reduction from baseline values. At 1 year, 27% patients had the same NCS as baseline, 32% showed a reduction by 1, 21% reduced by 2, and 5% showed a reduction by 3 in the total score. The angle of deviation with the overminus for both distance and near also showed significant reduction. Conclusion: This study showed definite improvement in control of IXT with overminus, and hence, a trial of overminus therapy is recommended for IXT in children below 5 years, as surgery has its own side effects like overcorrection, leading to consecutive esotropia with loss of stereopsis and high amount of recurrence

Osteocalcin Is Independently Associated with C-Reactive Protein during Lifestyle-Induced Weight Loss in Metabolic Syndrome

Bone-derived osteocalcin has been suggested to be a metabolic regulator. To scrutinize the relation between osteocalcin and peripheral insulin sensitivity, we analyzed changes in serum osteocalcin relative to changes in insulin sensitivity, low-grade inflammation, and bone mineral density following lifestyle-induced weight loss in individuals with metabolic syndrome (MetS). Participants with MetS were randomized to a weight loss program or to a control group. Before and after the 6-month intervention period, clinical and laboratory parameters and serum osteocalcin levels were determined. Changes in body composition were analyzed by dual-energy X-ray absorptiometry (DXA). In participants of the intervention group, weight loss resulted in improved insulin sensitivity and amelioration of inflammation. Increased serum levels of osteocalcin correlated inversely with BMI (r = −0.63; p < 0.001), total fat mass (r = −0.58, p < 0.001), total lean mass (r = −0.45, p < 0.001), C-reactive protein (CRP) (r = −0.37; p < 0.01), insulin (r = −0.4; p < 0.001), leptin (r = −0.53; p < 0.001), triglycerides (r = −0.42; p < 0.001), and alanine aminotransferase (ALAT) (r = −0.52; p < 0.001). Regression analysis revealed that osteocalcin was independently associated with changes in CRP but not with changes in insulin concentration, fat mass, or bone mineral density, suggesting that weight loss-induced higher serum osteocalcin is primarily associated with reduced inflammation