Engineers from UC San Diego have disclosed a new patent-pending technology (SHARP, VERTICALLY ALIGNED NANOWIRE ELECTRODE ARRAYS, HIGH-YIELD FABRICATION ANDINTRACELLULAR RECORDING) that minimizes the electrode size to an intracellular probe, and is scalable to integrate multiple channels at one platform and overcomes the previous disadvantages such as invasiveness and insensitivity. This newly disclosed improved technology reduces the number of steps and the number of metal layers used to increase the biocompatibility and device yield, as compared to an earlier disclosure for NEAs that were fabricated using a different process.

A major bottleneck in nanocarrier and macromolecule development for therapeutic delivery is our limited understanding of the processes involved in their uptake into target cells. This includes their active interactions with membrane transporters that co-ordinate cellular uptake and processing. Current strategies to elucidate the mechanism of uptake, such as painstaking manipulation of individual effectors with pharmacological inhibitors or specific genetic knockdowns, are limited in scope and biased towards previously studied pathways or the intuition of the investigators. Furthermore, each of these approaches present significant off-target effects, clouding the outcomes. Methods for intracellular transport of nucleic acids are much sought after in the context of both in vitro delivery reagents and in vivo therapeutics. Recently, we found that micellar assemblies of hundreds of amphiphiles consisting of single-stranded DNA which has been covalently linked to a hydrophobic polymer, referred to as DNA-polymer amphiphile nanoparticles or DPANPs, can readily access the cytosol of cells where they modulate mRNA expression of target genomes without transfection or other helper reagents, making them potential therapeutic nucleic acid carriers. However, despite their effective uptake properties and efficacy in the cytosol, it was unknown how these polyanionic structures can enter cells. Indeed, generally, bottlenecks in understanding and achieving delivery and uptake remain a forefront issue in translatability of macromolecular and nanomaterials-based therapeutics generally, including with respect to nucleic acid therapies. The nature of pooled screening requires amplifying a single ~200nt region per cell, leading to screens that require amplification from tens-to hundreds of micrograms of genomic DNA. Inhibitory effects of high DNA concentration per PCR have led to a variety of solutions, ranging from simply pooling hundreds of PCR reactions to utilizing restriction enzyme sites present in the lentiviral backbone constant regions flanking the sgRNA to perform DNA gel electrophoresis and size selection to remove undesired gDNA. However, these approaches can be both expensive and have significant handling challenges when scaled to large screens.

Cellular lipid membranes are embedded with transmembrane proteins crucial to cell function. Elucidating membrane proteins’ diverse structures and biophysical mechanisms is increasingly necessary due to their growing prevalence as a therapeutic target and sheer ubiquity in cells. Most biophysical characterization strategies of transmembrane proteins rely on the tedious overexpression and isolation of recombinant proteins and their reconstitution in model phospholipid bilayers.Unfortunately, membrane protein reconstitution depends on the use of denaturing and unnatural detergents that can interfere with protein structure and function. We have developed a detergent‐free method to reconstitute transmembrane proteins in model phospholipid vesicles and GUVs. Additionally, transmembrane proteins are difficult to express in cells due to the extreme insolubility of their transmembrane domain. By incorporating a synthetic transmembrane peptide into liposomes and simply expressing soluble portions of transmembrane proteins in cells, we can use this semisynthetic ligation strategy to more easily construct functional transmembrane proteins and reconstitute them into liposomes for biophysical and biochemical studies.Inteins can be found contiguously or non contiguously within some proteins. Non‐contiguous inteins are called “split inteins”. Inteins can be thought of as a type of protein intron which splices itself out of proteins. When non‐contiguous inteins find and bind to each other, they are then able to excise themselves resulting in the ligation of their respective exteins. Split intein pairs (C‐intein and N‐intein) can be attached to proteins of interest in synthetic and cellular systems to ligate protein sequences together.

Beta-lactam antibiotics account for the majority of antibiotics used worldwide. Resistance by beta-lactamase expression is a serious and growing threat. The typical workflow in a clinical microbiology laboratory leading to identification of antibiotic resistant organisms consists of 1) sample plating and mixed growth, 2) pathogen isolation and growth, 3) identification of the organism by biochemical tests or  Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF), and finally 4) observed growth in antibiotic containing media to determine antibiotic susceptibility/resistance patterns. This workflow requires 36 to 72 hours, involves multiple manual steps, and may not detect inducible resistance. The evolution and spread of antibiotic resistance among human pathogens represents a serious public health threat. Faster identification of the presence of antibiotic resistant organisms is a key component in the effort to reduce the spread of antibiotic resistance, as evidenced by the inclusion of diagnostic development in the CDC’s national strategy to combat antibiotic resistance. Given the clinical challenges that beta-lactamase expressing pathogens present, there is a clear need for faster identification to both enable effective treatment and to enact isolation precautions preventing further spread of resistant organisms Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} RNA binding proteins are increasingly implicated in genetic and somatic diseases.  Higher resolution methods to identify their RNA targets and how the proteins may interact with specific bases within them are needed to develop drugs that interfere with the regulation or misregulation of RBPs via their binding sites.

Inflammatory bowel disease is a chronic disease, which affects the lower bowel parts or the entire GI tract, causing symptoms like abdominal pain, diarrhea, fever and weight loss. An estimated two million people in North America suffer from IBD seemingly caused by an overactive mucosal immune system. Crohn’s Disease and ulcerative colitis (UC) are the major groups of inflammatory conditions that make up IBD and are incurable, serious and chronic organic diseases of the intestinal tract.   Recently, anti-integrin monoclonal antibodies have been approved by the FDA as therapeutic agents for treatment of IBD and there are a number of phase three clinical trials ongoing using monoclonal antibody therapy. The immune system responds to the inflammation that is part of the immunopathology of IBD and acts by recruiting inflammatory cells to the intestinal lesions.  Intergrins, specifically alpha 4-β7, plays a key role in mediating leukocyte trafficking from the circulation to the vascular endothelial barrier in gut-associate lymphoid tissue with the ligand MAdCAM-1. The use of anti-integrin therapy targeting alpha 4-β7 reduces the number of immune cells to the gut endothelium. However, the precise identity of the cell subsets depleted from the intestinal lamina by these anti-integrin drugs have not been identified. Thus, there is an unmet need to further develop tools that allow for the identification of the critical effector cell subsets targeted by these drugs in the intestine.

Cardiovascular disease is the leading cause of mortality and disability worldwide. It is also prevalent, affecting 9% of the population over 20 years of age. Patients with cardiovascular risk factors can reduce their risk of developing catastrophic cardiovascular events such as heart attack and stroke through lifestyle modification and medications. Unfortunately for many, the disease may go undiagnosed until the occurrence of serious events. Identifying biomarkers of subclinical ischemia can help identify patients with occult cardiovascular disease.

Modeling diseases as networks has helped simplify an otherwise complex web of multi‐cellular processes; however, an exclusive reliance on symmetric relationships in these networks overlooks the existence of disease continuum states and loses information relevant to pathogenesis and for the development of therapeutics. Network‐based analyses severely influenced by symmetric analyses have helped formalize Network Medicine as a field and deliver many successes, but drugs that can predictably re‐set the network in complex multi‐component diseases are yet to emerge.

Uveal melanoma commonly known as ocular or choroidal melanoma, is a rare cancer of the eye. It is an intraocular malignancy that arises from melanocytes of the choroid, ciliary body, and iris of the eye. Ocular melanoma is diagnosed in approximately 2,000-2,500 adults annually in the United States. In both the U.S. and Europe, this equates to about 5 - 7.5 cases per million people per year and, for people over 50 years old, the incidence rate increases to around 21 per million per year. While the primary tumor is highly treatable, about half of the patients will develop metastasis —typically to the liver. Metastatic disease is universally fatal. While traditional staging methods such as tumor size and location, still play a role in assessing metastatic risk, they are rarely used to individualize patient management plans. Newer methods include chromosomal gene expression analysis, yet these methods have their technical limitations. Clearly, what is needed is a better, cheaper and reproducible prognostic test.

A mosaic of cross-phyla chemical interactions occurs between all metazoans and their microbiomes. In humans, the gut harbors the heaviest microbial load, but many organs, particularly those with a mucosal surface, associate with highly adapted and evolved microbial consortia. The microbial residents within these organ systems are increasingly well characterized, yielding a good understanding of human microbiome composition. However, we have yet to elucidate the full chemical impact the microbiome exerts on an animal and the breadth of the chemical diversity it contributes. A number of molecular families are known to be shaped by the microbiome including short-chain fatty acids, indoles, aromatic amino acid metabolites, complex polysaccharides, and host sphingolipids and bile acids. These metabolites profoundly affect host physiology and are being explored for their roles in both health and disease. The synthesis of bile acids takes place in the liver and recent research has shown that bile acids can act as signaling molecules and activate a number of molecules. A primary focus has been on the Farnesoid X receptor (FXR) which plays an important role in bile acid synthesis and in regulation of glucose, lipid and energy metabolism.

Vedolizumab (VDZ) is an effective therapy for the management of patients with moderately to severely active ulcerative colitis (UC) or Crohn’s disease (CD) who have failed conventional therapy with aminosalicylates, corticosteroids, and thiopurines, as well as biologic therapy with tumor necrosis factor (TNF) antagonists. Several studies have identified potential predictors of treatment outcomes; however, the optimal approach to integrating predictors into routine practice is uncertain.No prior decision support tools exist to predict VDZ drug exposure in UC and CD and link this back to differences in effectiveness or response to VDZ dose escalation. By having a tool that can predict at baseline prior to start of therapy whether VDZ will be effective and what a patients drug exposure profile will be with VDZ, the provider can 1) determine if VDZ is an appropriate therapy to begin, 2) proactively monitor those patients deemed high risk for treatment failure with VDZ, and 3) proactively measure drug concentrations for VDZ to then increase the dose or the interval at which VDZ is administered to improve outcomes.

Routine clinical evaluation of osteoporosis (OP) has been focused on dual energy X-ray absorptiometry(DEXA) and/or computed tomography (CT), which provides qualitative analysis of bone mineral (~45% of bone by volume). The majority of bone which is the organic matrix and water (~55% of bone by volume) plays an important role in bone viscosity and strength. Bone mineral density (BMD) by itself only predicts fractures with an accuracy of 30-50%. The overall fracture risk increases 13-fold from ages 60 to 80, but BMD alone only predicts a doubling of the fracture risk. A recent study of over 7806 patients found that only 44% of all non-vertebral fractures occurred in women with a T-score below -2.5 (WHO definition of OP). This percentage dropped to 21% in men. There is a clear need for more sensitive risk assessment tools which not only use BMD, but other determinants of risk such as bone microstructure, porosity, organic matrix and bone water. The organic matrix and water are undetectable with any of the current non-invasive imaging and/or quantification techniques. Magnetic resonance imaging (MRI) detects signals from water in tissues, thus potential for detecting the collagen matrix (bound water) and bone porosity (bulk water). However, bone water has very short transverse relaxation time (T2*) and is undetectable using conventional MR sequences on clinical MR systems.

In general, the risk of having a child with autism spectrum disorder (ASD) is about 1 in 68, or 1.5%. But the risk goes up for families who already have a child with ASD. If a family has one child with ASD, the chance of the next child having ASD is about 20%. If the next child is a boy, the risk is 26%, whereas if it’s a girl the risk is 10%. About 47% of families had more than one child with autism. Currently if a child has a birth defect or autism, the emerging trend is to perform whole exome sequencing to identify genetic mutations. These mutations overwhelmingly come from the father, because sperm cells but not egg cells continue to divide through the life of adults. Once the mutation is identified, the diagnosis can be made in the child, but the parents are left wondering if this genetic event could recur in future children. Currently there is no genetic assessment of sperm available commercially, and no publications on the application of using sperm as a way to assess risk of childhood disease, nor is there a risk assessment available for couples that have had a child with a genetic disease due to de novo genetic mutation.

The routine cancer screening tests are mainly lengthy processes, which can be invasive and costly. Blood-based screening is an attractive option, as routine medical visits offer the opportunity to collect blood samples that may be screened for signs of disease. Currently the available options are limited.

  Heparin anticoagulation therapy is a cornerstone of surgical and cardiovascular medicine because of its short half-life, reversible nature, and low cost, but it also suffers from a narrow therapeutic window and is the second most common medication error. It is used prophylactically in pediatric patients undergoing angiography, bypass, cannulation, extracorporeal membrane oxygenation, as well as therapeutically in thromboses, and cancer. Heparin is used in 15% of pediatric inpatients, and approximately one in seven patients has an adverse event, for an annual morbidity of 140,000; mortality rates of the diseases treated with heparin are 2-20% and include cerebral sinovenous thrombosis and vascular thromboembolism. These inherent challenges are compounded by iatrogenic errors such as incorrect heparin ordering, inaccurate infusion pump settings, and incomplete record keeping, resulting in ~10,000 heparin medication errors per year. Heparin is involved in more medication errors than morphine and vancomycin with multiple deaths annually particularly in neonates. Thousands more suffer from hemorrhages or emboli because of inaccurate dosing and dosing/monitoring programs that are designed for adult populations. Because of these issues, heparin anticoagulation therapy must be monitored very carefully. The current standard of care is the activated partial thromboplastin time; however, this in vitro diagnostic tool requires large blood volumes and suffers from long turnaround times, a variable pediatric reference range, and poor correlation to heparin dose/patient performance. Clearly, there is an unmet need for a real-time and non-invasive tool to monitor heparin.1The field of pediatric coagulation are desperate for new and improved analytical instrumentation to increase the efficacy of heparin monitoring.  

The researcher has developed a novel fluorescent reporter mouse in which fluorescent signals reflected endogenous Msi expression (Msi1eYFP, Msi2eGFP, Msi1 reporter mice (Reporter for Musashi1, or REM1) showed expression in the stem cell enriched adult subventricular zone, and Msi1+ cells were Nestin+ and CD133+  consistent with Msi1 marking neural stem/progenitor cells. Msi2 reporters (REM2) reflected endogenous expression of Msi2, being  highest in hematopoietic stem cells and declining with maturation.The Msi reporters described here represent exciting new tools that could be broadly useful for studying cancer. Because Msi reporter activity can be visualized through live imaging these reporter mice can be uniquely used to image and track cancer stem cells in vivo, and can provide a dynamic view of endogenous cancer growth, tumor dissemination and metastasis in its native microenvironment.  The fact that reporter positive cells are preferentially gemcitabine resistant, raises the exciting possibility that this could serve as a new platform to identify therapy resistance in vivo. The integration of such reporters in drug development may provide a powerful and sophisticated complement to traditional screens, by allowing the identification of therapies that are better able to target tumor propagating cells, and drug resistant residual disease. In addition, the spatially restricted distribution of Msi+ cells could have important implications for loco regional, aggressive targeting of driver cells that mediate resistance and disease relapse.

1-D electrophysiological probes of the needle and micropipette configuration were the first tools to measure action potentials in neurons.   Since its development in the 1970s, this patch-clamp technique remains the standard in high fidelity detection of small potential subthreshold activity.  However, the use of tapered submicron micropipette tips to patch into cell membranes and measure sub-threshold potentials and ionic currents are not scalable to large neuronal densities and to long recording times. Automated patch-clamp are scalable but cannot perform recordings from networks of neurons that resemble cell arrangements in organs from brain, to heart, to muscle, to liver, etc.  Microelectrode arrays (MEAs) on the other hand are scalable but their planar geometry preclude intimate interaction and sufficient charge coupling between the neuron and the electrode site.  The weakly coupled sub-threshold activity in MEAs is usually below the noise level and is therefore lost and not observed in MEA measurements. None of prior technologies, can sense activity in 3D networks of neurons. Nanowire geometries are ideal for minimally invasive intracellular nanoscale probes but prior works have been limited to single nanowire device demonstrations or to devices encompassing ensembles of several nanowires and without sensitivity to subthreshold neuron activity or demonstration of interfacing with human neurons. 

Cancer relapse and subsequent metastatic disease pose the most critical challenges in anticancer drug discovery and development. The limited clinical translational value of analyses performed on 2D cell cultures has prompted a shift toward the generation of 3-dimensional (3D) multicellular systems. One such 3D model system is the spontaneously-forming in vitro cancer spheroid model, referred to as spheroidsMARY-X, which precisely reflects the pathophysiological features of both tumor tissue and the lymphovascular embolus. However, these spheroids were not previously compatible with options for commercial high throughput drug screening.

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Cancer invasion/metastasis, diabetes, cardiac fibrosis, schizophrenia etc are all multigenetic diseases that is the end result of an aberrant signalling network, usually multi-receptor in etiology. To halt/reverse these progressive diseases it will take more than just blocking one receptor/pathway at a time. Fundamentally, the development of silver-bullet therapies to treat these conditions needs identification and targeting of molecular hubs that can modulate incoming aberrant signals from multiple receptors. We propose to target such a powerful hub, a unique protein-protein interface, which is dysregulated in each of these conditions, and primarily serves to modulate (amplify or attenuate) pathogenic signal networks triggered by multiple receptors. Targeting such hubs with agonists or antagonists will serve as tools for shifting the aberrant network in diseased cells back to a stable, physiological pattern. This is extremely important because most network-based therapies will help reshape the entire signaling network so that it lies in a new and stable region of behavior space.

A group of blood biomarkers has been identified for the diagnosis of complications due to cirrhosis. They correlate with the presence or absence of clinically significant increased portal pressure characteristic of chronic liver diseases. The clinically significant level of increased portal pressure is a required threshold for the presence of esophageal varices and their potential for inducing internal bleeding, a major complication in patients with chronic liver diseases.There are approximately 200 million people with Chronic Hepatitis C infection worldwide; 1 million cirrhosis patients admitted to hospitals in Europe and the USA; 4 million with Hepatitis B or C in the USA; 10 million with non-alcoholic steatohepatitis (NASH) related to obesity and Diabetes type 2 in the USA. These chronic liver diseases may develop clinically significant portal hypertension with risk of developing esophageal varices.At present the Standard of Care Guidelines for cirrhosis patients from the American Association for the Study of Liver Diseases recommends Esophagogastroduodenoscopy (‘Upper Endoscopy’) yearly or every other year.The approximate cost of ‘Upper Endoscopy’ is currently $10,000 and the procedure is not without risk to the patient, and has been associated with significant adverse events (respiratory depression; internal bleeding and infections). All of the above patients would benefit from this biomarker screening, as well as any other patient presenting with previously undiagnosed liver symptoms. This biomarker panel would be prescribed for any diagnosed liver patient or any patient presenting with possible liver symptoms to enable clinical decisions to be made for patient care in a medically and financially expeditious manner.

CLL is the most common form of adult leukemia in the Western world. It is characterized by the accumulation of CD5+, CD 19+ and CD23+ B-cells due to decreased apoptosis. CLL shows a highly variable clinical course spanning from indolent, slow growing, to aggressive, which requires immediate treatment. A clinical problem for many heterogeneous diseases, such as chronic lymphocytic leukemia, is the lack of identification of molecular and cellular markers that can predict progression. G protein-coupled receptors (GPCR) are guanine nucleotide exchange factors for heterotrimeric G-proteins, whose α and βy subunits dissociate and regulate effectors. Gαs stimulates adenylyl cyclase, and Gαi inhibits adenylyl cyclase. GPCRs are the largest receptor family (-3% of genome) and are the largest class of attractive drug targets in disease since they are expressed on the plasma membrane and are tissue specific.

Mutations and duplication of the X-linked MeCP2 gene are observed in several disorders, such as Rett Syndrome (RTT), Autism, severe neonatal encephalopathy, schizophrenia and X-linked mental retardation. As MeCP2 plays an important role in the pathogenesis of multiple mental disorders, the investigation of MeCP2 function and regulatory pathways may show promise for developing broad-spectrum therapies. Downstream MeCP2 target genes, such as the calcium channel TRPC6, can have a secondary impact on the cell and predispose the individual to autism.

The ability to capture and study circulating tumor cells is an emerging field with implications for early detection, diagnosis, determining prognosis, and monitoring of cancer, as well as for understanding the fundamental biology of metastasis. Current techniques of identifying and isolating such cells usually involve flowing cells in a chip across an antibody coated surface. However, these devices usually require complex geometries to ensure effective contact of the target cells with the functionalized surfaces. Such a problem can be avoided by using micro/nanoscale motors that can be programmed to scower an entire static sample as many times as needed. Further, the movement of the nano/microscale motor increases the solution convection thereby improving the diffusion of the target antigen, making for a quicker and more favorable recognition reaction. This also helps eliminate non-specific binding of the antigen while on its way to a clean environment for post-capture analysis.

Electrochemical DNA biosensors are simple, inexpensive, and portable, making them attractive for decentralized genetic testing. Surface chemistry plays a major role in the overall performance of such biosensors. In particular, surface chemistry and coverage control is essential for assuring high reactivity, orientation/accessibility, and stability, while avoiding nonspecific adsorption and related background contributions. Several schemes for attaching nucleic acid probes to electrode surfaces and controlling the surface chemistry have thus been developed. Alkanethiol self-assembled monolayer (SAM) methods have been particularly useful for preparing reproducible probe-modified surfaces with high hybridization efficiency. Most often, two-component SAM monolayers of thiol-derivatized single-stranded oligonucleotide probe (thiolated capture probe, SHCP) and a short-chain 6-mercapto-1-hexanol (MCH) are used. Yet, such binary monolayers still suffer from background contributions and irreproducibility problems resulting from incomplete backfilling and surface defects.