The burden of severe hypercholesterolemia and familial hypercholesterolemia in a population-based setting in the US.

Background: Contemporary prevalence, awareness, and control of severe hypercholesterolemia (SH) and familial hypercholesterolemia (FH) and the associated atherosclerotic cardiovascular disease risk in the US are unknown. Method: Using electronic health records, we assessed the burden of SH and FH in Olmsted County, Minnesota, US, between 2004 and 2015. We defined SH as low-density lipoprotein cholesterol (LDL-C) level ≥190 mg/dl without secondary causes of hypercholesterolemia and FH as a Dutch Lipid Clinic Network score ≥6. Controls were age- and sex-matched individuals with LDL-C level <190 mg/dl. Results: The age- and sex-adjusted point and period prevalence (age-recursive method) of SH was 4.44% and 8.95%, respectively; 1 in 21 had FH (∼1:233 adults), and 46.2% had a recorded diagnosis. Guideline recommended targets (LDL-C <100 mg/dl and <70 mg/dl in the primary and secondary prevention settings, respectively) were achieved in 33.1% and 21.2% of SH cases, with less women overall achieving the target than men (18.6% vs. 23.7%, p=0.022). After adjustment for conventional risk factors, the hazard ratio for incident coronary heart disease (CHD) in those with SH was 1.21 (1.05-1.39; p=0.010), in those with SH and a family history of CHD was 2.16 (1.57-2.96; p<0.001) and in those with FH was 4.61 (2.66-7.97; p<0.001). The association of SH with CHD was modified by age (p-interaction = 0.015), such that the risk was greater at younger ages. Conclusions: SH was prevalent and an independent risk factor for incident CHD. Awareness and control were low, highlighting a treatment gap (more prominent in women) that needs to be addressed.

Flagellar rotational features of an optically confined bacterium at high frequency and temporal resolution reveal the microorganism's response to changes in the fluid environment.

Rotations of the flagella control the movement of a peritrichous (multiflagellar) bacterium in fluids, the run and tumble events being caused through modulations in the flagella's collective rotation speed and pattern. Observing such modulations is a challenge in free swimming bacteria. In this work, we present a setup to measure the collective flagellar rotational features of an optically confined Bacillus subtilis bacterium. We adopt a Continuous Wavelet Technique (CWT) while monitoring the rotational patterns in frequency and time, thus achieving optimal resolution in both the domains. This enables in marking the events wherein subtle changes in the flagellar rotational pattern occur. These studies unravel a fact, hitherto unknown, that variations in swimming speed that are seen in pure run sequences are also caused by modulations in the rotating flagella. Further, we have monitored the flagellar rotation for durations over a minute and observe a gradual slowing down of the rotation before ceasing completely due to the trapping laser induced photodamage. We have observed a significant alteration in the rate of rotational fall off in real time with changes in pH or the nutrient concentration in the fluid. This work serves to demonstrate the advantage of optical confinement of a bacterium in its pristine form for carrying out such studies and can serve as a marker for work that assesses membrane photodamage in active matter. Details on the role of flagella in propulsion and on other factors influencing the rotations, can be of significance in the design of artificial microswimmers.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array.

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin-polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.

A Case of Reverse Palmaris Longus Muscle- An Additional Muscle in the Anterior Compartment of the Forearm.

It is uncommon to have additional muscles in the upper limb. Some of them may restrict the movements or compress the nerves and vessels, while others may go unnoticed. During the routine dissection for undergraduate medical students, we observed an additional muscle in the anterior compartment of the forearm in about 60-year-old male cadaver. The muscle had a prominent belly and a long tendon. Distally, it was attached to the flexor retinaculum by a short and thick tendon. Proximally, long tendon of the muscle passed between the flexor carpi ulnaris and palmaris longus and was attached to the common aponeurosis shared by the extensor carpi ulnaris and flexor digitorum profundus muscles. The additional muscle belly was supplied by a branch from the anterior interosseous nerve. The ulnar nerve and artery was passing deep to the fleshy belly of the muscle. The muscle reported here might compress the ulnar nerve and artery and may produce neurovascular symptoms. On the other hand, the tendon and fleshy belly of the muscle could be useful in muscle/tendon grafts. The observations made by us in the present case will supplement our knowledge of variations of the muscles in this region which could be useful for surgeons during the forearm and hand surgeries.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Ohtahara syndrome associated with hemimegalencephaly and intracranial lipoma.

Hemimegalencephaly is a disorder of cortical malformation and is associated with various disorders including various neurocutaneous syndromes and many seizure types. We present a case of hemimegalencephaly associated with Ohtahara syndrome and intracranial and facial lipoma.

Systems level mapping of metabolic complexity in Mycobacterium tuberculosis to identify high-value drug targets.

BACKGROUND: The effectiveness of current therapeutic regimens for Mycobacterium tuberculosis (Mtb) is diminished by the need for prolonged therapy and the rise of drug resistant/tolerant strains. This global health threat, despite decades of basic research and a wealth of legacy knowledge, is due to a lack of systems level understanding that can innovate the process of fast acting and high efficacy drug discovery. METHODS: The enhanced functional annotations of the Mtb genome, which were previously obtained through a crowd sourcing approach was used to reconstruct the metabolic network of Mtb in a bottom up manner. We represent this information by developing a novel Systems Biology Spindle Map of Metabolism (SBSM) and comprehend its static and dynamic structure using various computational approaches based on simulation and design. RESULTS: The reconstructed metabolism of Mtb encompasses 961 metabolites, involved in 1152 reactions catalyzed by 890 protein coding genes, organized into 50 pathways. By accounting for static and dynamic analysis of SBSM in Mtb we identified various critical proteins required for the growth and survival of bacteria. Further, we assessed the potential of these proteins as putative drug targets that are fast acting and less toxic. Further, we formulate a novel concept of metabolic persister genes (MPGs) and compared our predictions with published in vitro and in vivo experimental evidence. Through such analyses, we report for the first time that de novo biosynthesis of NAD may give rise to bacterial persistence in Mtb under conditions of metabolic stress induced by conventional anti-tuberculosis therapy. We propose such MPG's as potential combination of drug targets for existing antibiotics that can improve their efficacy and efficiency for drug tolerant bacteria. CONCLUSION: The systems level framework formulated by us to identify potential non-toxic drug targets and strategies to circumvent the issue of bacterial persistence can substantially aid in the process of TB drug discovery and translational research.

Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.

A systems perspective of host-pathogen interactions: predicting disease outcome in tuberculosis.

The complex web of interactions between the host immune system and the pathogen determines the outcome of any infection. A computational model of this interaction network, which encodes complex interplay among host and bacterial components, forms a useful basis for improving the understanding of pathogenesis, in filling knowledge gaps and consequently to identify strategies to counter the disease. We have built an extensive model of the Mycobacterium tuberculosis host-pathogen interactome, consisting of 75 nodes corresponding to host and pathogen molecules, cells, cellular states or processes. Vaccination effects, clearance efficiencies due to drugs and growth rates have also been encoded in the model. The system is modelled as a Boolean network. Virtual deletion experiments, multiple parameter scans and analysis of the system's response to perturbations, indicate that disabling processes such as phagocytosis and phagolysosome fusion or cytokines such as TNF-alpha and IFN-gamma, greatly impaired bacterial clearance, while removing cytokines such as IL-10 alongside bacterial defence proteins such as SapM greatly favour clearance. Simulations indicate a high propensity of the pathogen to persist under different conditions.

Modeling metabolic adjustment in Mycobacterium tuberculosis upon treatment with isoniazid.

UNLABELLED: Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9075-6) contains supplementary material, which is available to authorized users.