Magnetically coupled percutaneous connector for chronic electrical peripheral nerve stimulation and recording in awake rats.

A fast-growing field of neuroscience and medicine is the treatment of disease via electrical stimulation of the peripheral nervous system. Peripheral nerve stimulation delivers stimulation to nerves of the periphery where the target nerve can and is often located deep within the abdomen. Long-term preclinical animal models that demonstrate the safety and/or efficacy of electrical stimulation have predominantly used a skull mount to connect to neural interfaces. When targeting nerves of the extremities and abdomen, this mount location is less favourable due to its distance to the implant causing complications in surgery and to the longevity of the device in vivo. OBJECTIVE: Here we aimed to develop and validate a chronic magnetic percutaneous connector designed for placement on the dorsal-lumbar aspect of the spine of awake, freely moving rats. METHODS: A pedestal and external connector was developed, bench tested to assess for continuity, durability and disconnection forces, and validated in awake rats chronically implanted with an abdominal vagus nerve electrode array. The implanted pedestal and external connector were designed with custom PCBs, spring-loaded pins, magnets and biocompatible 3D printed housing. RESULTS: The magnetic coupling mechanism allowed disconnection with minimal force, was highly reliable in maintaining electrical connection in awake rats and allowed recording of electrically evoked compound action potentials after chronic implantation. CONCLUSION: In conclusion, this percutaneous connector is a useful research tool for peripheral nerve stimulation studies. SIGNIFICANCE: The connector described will allow investigation into the safety and efficacy of emerging neuromodulation therapies for the treatment of disease.

Exploring the role of casein kinase 1α splice variants across cancer cell lines.

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.

Triptolide and its prodrug Minnelide target high-risk MYC-amplified medulloblastoma in preclinical models.

Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic data set that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor 5-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB.

Twenty Years of Subcutaneous Interferon-Beta-1a for Multiple Sclerosis: Contemporary Perspectives.

Multiple sclerosis (MS) is a chronic, progressive, inflammatory disorder of the central nervous system. Relapsing-remitting MS (RRMS), the most common form of the disease, is characterized by transient neurological dysfunction with concurrent accumulation of disability. Over the past three decades, disease-modifying therapies (DMTs) capable of reducing the frequency of relapses and slowing disability worsening have been studied and approved for use in patients with RRMS. The first DMTs were interferon-betas (IFN-ßs), which were approved in the 1990s. Among them was IFN-ß-1a for subcutaneous (sc) injection (Rebif®), which was approved for the treatment of MS in Europe and Canada in 1998 and in the USA in 2002. Twenty years of clinical data and experience have supported the efficacy and safety of IFN-ß-1a sc in the treatment of RRMS, including pivotal trials, real-world data, and extension studies lasting up to 15 years past initial treatment. Today, IFN-ß-1a sc remains an important therapeutic option in clinical use, especially around pregnancy planning and lactation, and may also be considered for aging patients, in which MS activity declines and long-term immunosuppression associated with some alternative therapies is a concern. In addition, IFN-ß-1a sc is used as a comparator in many clinical studies and provides a framework for research into the mechanisms by which MS begins and progresses.

Effectiveness and safety of switching to teriflunomide in older patients with relapsing multiple sclerosis: A real-world retrospective multicenter analysis.

BACKGROUND: The prevalence of multiple sclerosis (MS) in older people is increasing due to population aging and availability of effective disease-modifying therapies (DMTs). Treating older people with MS is complicated by age-related and MS-related comorbidities, immunologic effects of prior DMTs, and immunosenescence. Teriflunomide is a once-daily oral immunomodulator that has demonstrated efficacy and acceptable safety in clinical trials of adults with relapsing forms of MS (RMS). However, there are limited clinical trial and real-world data regarding teriflunomide use in people with MS aged >55 years. We analyzed real-world data to assess the effectiveness and safety of teriflunomide in older people with RMS who had switched to this agent from other DMTs. METHODS: People with RMS (relapsing remitting and active secondary progressive MS) aged ≥55 years who had switched from other DMTs to teriflunomide (7 mg or 14 mg) for ≥1 year were identified retrospectively by chart review at four sites in the United States. Data were extracted from medical records from 1 year pre-index to 2 years post-index (index defined as the teriflunomide start date). Assessments of effectiveness included annualized relapse rate (ARR), Expanded Disability Status Scale (EDSS) score, and magnetic resonance imaging (MRI) outcomes. Assessments of safety included lymphocyte counts, infections, and malignancies. We examined the effectiveness outcomes and lymphocyte counts within sub-groups defined by age (55-64, ≥65 years), sex, MS type, and prior route of DMT administration (oral, injectable, infusible). RESULTS: In total, 182 patients with RMS aged ≥55 years who switched from other DMTs to teriflunomide were identified (mean [SD] age: 62.5 [5.4] years). Mean ARR decreased from the start of teriflunomide treatment (mean [SD]: 0.43 [0.61]) to year 1 post-index (0.13 [0.65]) and year 2 post-index (0.05 [0.28]). Mean EDSS score remained unchanged from index (mean [SD]: 4.5 [1.8]) to 1 year post-treatment (4.5 [1.8]) and increased slightly at 2 years post-treatment (4.7 [1.7]). MRI scans from index and years 1 and 2 post-index compared with scans from the previous year indicated that most patients had stable or improved MRI outcomes at index (87.7%) and remained stable or improved at years 1 (96.0%) and 2 (93.6%). Lymphopenia decreased at years 1 (21.4%) and 2 post-index (14.8%, compared to index (23.5%). By 1 year post-index, fewer patients had grade 3 or 4 lymphopenia, and at 2 years post-index, there were no patients with grade 3 or 4 lymphopenia. Infection incidence was low (n = 40, 22.0%) and none were related to teriflunomide. The decreases in lymphopenia were driven by decreases among people who switched from a prior oral DMT; there were no notable differences in lymphopenia across the other sub-groups examined. ARR, EDSS score, and MRI outcomes across all sub-groups were similar to the results of the overall population. CONCLUSION: Our multicenter, longitudinal, retrospective study demonstrated that patients with RMS aged 55 or older switching to teriflunomide from other DMTs had significantly improved ARR, stable disability, and stable or improved MRI over up to 2 years' follow up. Safety results were acceptable with fewer patients exhibiting lymphopenia at years 1 and 2 post-index.

Multiprotein GLI Transcriptional Complexes as Therapeutic Targets in Cancer.

The Hedgehog signaling pathway functions in both embryonic development and adult tissue homeostasis. Importantly, its aberrant activation is also implicated in the progression of multiple types of cancer, including basal cell carcinoma and medulloblastoma. GLI transcription factors function as the ultimate effectors of the Hedgehog signaling pathway. Their activity is regulated by this signaling cascade via their mRNA expression, protein stability, subcellular localization, and ultimately their transcriptional activity. Further, GLI proteins are also regulated by a variety of non-canonical mechanisms in addition to the canonical Hedgehog pathway. Recently, with an increased understanding of epigenetic gene regulation, novel transcriptional regulators have been identified that interact with GLI proteins in multi-protein complexes to regulate GLI transcriptional activity. Such complexes have added another layer of complexity to the regulation of GLI proteins. Here, we summarize recent work on the regulation of GLI transcriptional activity by these novel protein complexes and describe their relevance to cancer, as such GLI regulators represent alternative and innovative druggable targets in GLI-dependent cancers.

Protein arginine methyltransferase 5 regulates SHH-subgroup medulloblastoma progression.

Background: Medulloblastoma (MB) is the most common pediatric brain tumor. Although standard-of-care treatment generally results in good prognosis, many patients exhibit treatment-associated lifelong disabilities. This outcome could be improved by employing therapies targeting the molecular drivers of this cancer. Attempts to do so in the SONIC HEDGEHOG MB subgroup (SHH-MB) have largely focused on the SHH pathway's principal activator, smoothened (SMO). While inhibitors targeting SMO have shown clinical efficacy, recurrence and resistance are frequently noted, likely resulting from mutations in or downstream of SMO. Therefore, identification of novel SHH regulators that act on the pathway's terminal effectors could be used to overcome or prevent such recurrence. We hypothesized that protein arginine methyltransferase 5 (PRMT5) is one such regulator and investigated its role and potential targeting in SHH-MB. Methods: PRMT5 expression in SHH-MB was first evaluated. Knockdown and pharmacological inhibitors of PRMT5 were used in SHH-MB sphere cultures to determine its effect on viability and SHH signaling. GLI1 arginine methylation was then characterized in primary SHH-MB tissue using LC-MS/MS. Finally, PRMT5 inhibitor efficacy was evaluated in vivo. Results: PRMT5 is overexpressed in SHH-MB tissue. Furthermore, SHH-MB viability and SHH activity is dependent on PRMT5. We found that GLI1 isolated from SHH-MB tissues is highly methylated, including three PRMT5 sites that affect SHH-MB cell viability. Importantly, tumor growth is decreased and survival increased in mice given PRMT5 inhibitor. Conclusions: PRMT5 is a requisite driver of SHH-MB that regulates tumor progression. A clinically relevant PRMT5 inhibitor represents a promising candidate drug for SHH-MB therapy.

Ublituximab versus Teriflunomide in Relapsing Multiple Sclerosis.

BACKGROUND: The monoclonal antibody ublituximab enhances antibody-dependent cellular cytolysis and produces B-cell depletion. Ublituximab is being evaluated for the treatment of relapsing multiple sclerosis. METHODS: In two identical, phase 3, double-blind, double-dummy trials (ULTIMATE I and II), participants with relapsing multiple sclerosis were randomly assigned in a 1:1 ratio to receive intravenous ublituximab (150 mg on day 1, followed by 450 mg on day 15 and at weeks 24, 48, and 72) and oral placebo or oral teriflunomide (14 mg once daily) and intravenous placebo. The primary end point was the annualized relapse rate. Secondary end points included the number of gadolinium-enhancing lesions on magnetic resonance imaging (MRI) by 96 weeks and worsening of disability. RESULTS: A total of 549 participants were enrolled in the ULTIMATE I trial, and 545 were enrolled in the ULTIMATE II trial; the median follow-up was 95 weeks. In the ULTIMATE I trial, the annualized relapse rate was 0.08 with ublituximab and 0.19 with teriflunomide (rate ratio, 0.41; 95% confidence interval [CI], 0.27 to 0.62; P<0.001); in the ULTIMATE II trial, the annualized relapse rate was 0.09 and 0.18, respectively (rate ratio, 0.51; 95% CI, 0.33 to 0.78; P = 0.002). The mean number of gadolinium-enhancing lesions was 0.02 in the ublituximab group and 0.49 in the teriflunomide group (rate ratio, 0.03; 95% CI, 0.02 to 0.06; P<0.001) in the ULTIMATE I trial and 0.01 and 0.25, respectively (rate ratio, 0.04; 95% CI, 0.02 to 0.06; P<0.001), in the ULTIMATE II trial. In the pooled analysis of the two trials, 5.2% of the participants in the ublituximab group and 5.9% in the teriflunomide group had worsening of disability at 12 weeks (hazard ratio, 0.84; 95% CI, 0.50 to 1.41; P = 0.51). Infusion-related reactions occurred in 47.7% of the participants in the ublituximab group. Serious infections occurred in 5.0% in the ublituximab group and in 2.9% in the teriflunomide group. CONCLUSIONS: Among participants with relapsing multiple sclerosis, ublituximab resulted in lower annualized relapse rates and fewer brain lesions on MRI than teriflunomide over a period of 96 weeks but did not result in a significantly lower risk of worsening of disability. Ublituximab was associated with infusion-related reactions. (Funded by TG Therapeutics; ULTIMATE I and II ClinicalTrials.gov numbers, NCT03277261 and NCT03277248.).

A Druggable UHRF1/DNMT1/GLI Complex Regulates Sonic Hedgehog-Dependent Tumor Growth.

Dysregulation of Sonic hedgehog (SHH) signaling drives the growth of distinct cancer subtypes, including medulloblastoma (MB). Such cancers have been treated in the clinic with a number of clinically relevant SHH inhibitors, the majority of which target the upstream SHH regulator, Smoothened (SMO). Despite considerable efficacy, many of these patients develop resistance to these drugs, primarily due to mutations in SMO. Therefore, it is essential to identify druggable, signaling components downstream of SMO to target in SMO inhibitor resistant cancers. We utilized an integrated functional genomics approach to identify epigenetic regulators of SHH signaling and identified a novel complex of Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), DNA methyltransferase 1 (DNMT1), and GLI proteins. We show that this complex is distinct from previously described UHRF1/DNMT1 complexes, suggesting that it works in concert to regulate GLI activity in SHH driven tumors. Importantly, we show that UHRF1/DNMT1/GLI complex stability is targeted by a repurposed FDA-approved therapy, with a subsequent reduction in the growth of SHH-dependent MB ex vivo and in vivo. IMPLICATIONS: This work describes a novel, druggable UHRF1/DNMT1/GLI complex that regulates SHH-dependent tumor growth, and highlights an FDA-approved drug capable of disrupting this complex to attenuate tumor growth.

Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.

Results from a multicenter, randomized, double-blind, placebo-controlled study of repository corticotropin injection for multiple sclerosis relapse that did not adequately respond to corticosteroids.

INTRODUCTION: About 20%-35% of multiple sclerosis (MS) patients fail to respond to high-dose corticosteroids during a relapse. Repository corticotropin injection (RCI, Acthar® Gel) is a naturally sourced complex mixture of adrenocorticotropic hormone analogs and pituitary peptides that has anti-inflammatory and immunomodulatory effects. AIMS: The study objective was to determine the efficacy and safety of RCI in patients with MS relapse that inadequately responded to corticosteroids. This was a multicenter, double-blind, placebo-controlled study. Nonresponders to high-dose corticosteroids were randomized to receive RCI (80 U) or placebo daily for 14 days. Assessments included improvements on the Expanded Disability Status Scale (EDSS), Multiple Sclerosis Impact Scale (MSIS-29), Clinical Global Impression of Improvement (CGI-I), and adverse events (AEs). RESULTS: Eighteen patients received RCI, and 17 received placebo. A greater proportion of EDSS responders was observed in the RCI group at Day 7, 21, and 42 compared with the placebo group. Qualitative CGI-I showed that more patients receiving RCI were much improved or very much improved than with placebo. No meaningful differences were observed between treatment groups for MSIS-29. No serious AEs or deaths were reported. CONCLUSION: RCI is safe and effective for MS relapse patients who do not respond to high-dose corticosteroids.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

Casein Kinase 1α as a Regulator of Wnt-Driven Cancer.

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.

Monomethyl fumarate has better gastrointestinal tolerability profile compared with dimethyl fumarate.

BACKGROUND: Monomethyl fumarate (MMF) is the pharmacologically active metabolite of dimethyl fumarate (DMF). MMF formulated as Bafiertam™ 190 mg and DMF formulated as Tecfidera 240 mg deliver bioequivalent exposure of MMF and therefore possess the same efficacy/safety profiles. DMF is a widely used oral treatment for relapsing-remitting forms of multiple sclerosis (RRMS) but is limited in some patients, primarily female, by issues with gastrointestinal (GI) tolerability. METHODS: This was a randomized, double-blind, head-to-head, 5-week study evaluating the GI tolerability of MMF 190 mg vs DMF 240 mg, administered twice daily in healthy subjects, using a derivative of the self-administered Modified Overall Gastrointestinal Symptom Scale (MOGISS). Subjects were stratified (3:1, female:male) and randomized (1:1) to the treatments. The primary endpoint was the Area Under the Curve (AUC) in each of the individual symptoms in the MOGISS over the 5-week treatment period. Other endpoints included the AUC over the 5-week treatment period in the MOGISS composite and total scores; duration and severity of GI events; Number and percentage of subjects reporting GI events during the overall treatment period, and assessment of safety/tolerability. RESULTS: Inferential analysis of the hierarchical testing of overall treatment differences in each MOGISS symptom AUC occurred in a predefined sequence starting with Abdominal Pain. For each symptom, LSMean AUC values were lower for MMF than DMF, however, the first primary endpoint, Abdominal Pain, was not statistically different between treatments; thus, all subsequent statistical analyses were considered exploratory. The side effects and safety profiles observed were consistent with the known profiles of DMF, with no new or unique safety concerns noted. CONCLUSIONS: Bafiertam showed an improved gastrointestinal tolerability profile compared with Tecfidera, with less severe GI events and fewer days of self-assessed GI symptoms, fewer GI adverse events, and lower discontinuation rates because of GI adverse events.

Incidence, management, and outcomes of autoimmune nephropathies following alemtuzumab treatment in patients with multiple sclerosis.

BACKGROUND: Autoimmune disorders including nephropathies have been reported more frequently in alemtuzumab-treated multiple sclerosis (MS) patients than in the general population. OBJECTIVE: Describe instances of autoimmune nephropathy in alemtuzumab-treated MS patients. METHODS: Cases were identified from safety monitoring within the alemtuzumab relapsing-remitting multiple sclerosis (RRMS) clinical development program (CDP) or post-marketing, or following off-label use. RESULTS: As of 16 June 2017, 16 autoimmune nephropathies have occurred following alemtuzumab treatment for MS. The incidence of autoimmune nephropathies was 0.34% within the CDP (5/1485 patients). The five CDP cases (one of anti-glomerular basement membrane (anti-GBM) disease, two of membranous glomerulonephropathy, and two of serum anti-GBM antibody without typical anti-GBM disease) were identified early, responded to conventional therapy (where needed), and had favorable outcomes. Three of 11 cases outside the CDP occurred following off-label alemtuzumab use prior to approval for RRMS and were all anti-GBM disease. Diagnosis was delayed in one of these three cases and another did not receive appropriate treatment; all three cases resulted in end-stage renal failure. All anti-GBM disease cases with documented urinalysis demonstrated prior microscopic hematuria. CONCLUSION: Close monitoring of alemtuzumab-treated MS patients facilitates diagnosis and treatment early in the nephropathy course when preservation of renal function is more likely.

Enduring Clinical Value of Copaxone® (Glatiramer Acetate) in Multiple Sclerosis after 20 Years of Use.

Multiple sclerosis (MS) is a chronic progressive neurodegenerative demyelinating disease affecting the central nervous system. Glatiramer acetate (GA; Copaxone®) was the first disease-modifying treatment (DMT) for MS successfully tested in humans (1977) and was approved by the US Food and Drug Administration in December 1996. Since then, there have been numerous developments in the MS field: advances in neuroimaging allowing more rapid and accurate diagnosis; the availability of a range of DMTs including immunosuppressant monoclonal antibodies and oral agents; a more holistic approach to treatment by multidisciplinary teams; and an improved awareness of the need to consider a patient's preferences and patient-reported outcomes such as quality of life. The use of GA has endured throughout these advances. The purpose of this article is to provide an overview of the important developments in the MS field during the 20 years since GA was approved and to review clinical data for GA in MS, with the aim of understanding the continued and widespread use of GA. Both drug-related (efficacy versus side-effect profile and monitoring requirements) and patient factors (preferences regarding mode of administration and possible pregnancy) will be explored.

Detection of bacterial contamination in food matrices by integration of quorum sensing in a paper-strip test.

There are an estimated 48 million cases of foodborne illness in the United States every year. In general, these illnesses are the result of unintentional contamination and improper food handling. Because bacterial contamination plays a major role in food spoilage and, hence, in foodborne illnesses, it is important to design easy, portable methods to detect bacteria in food. Quorum sensing (QS) enables bacteria to communicate with one another and by doing so they can modulate their behavior in a cell-density dependent manner. In bacteria, quorum sensing molecules (QSMs) are known to control several factors such as virulence factor production, antibiotic production, biofilm formation, and gene regulation. Herein, we demonstrate the applicability of whole cell biosensing systems for the early identification of food contamination via detection of QSMs. Additionally, we have developed a portable system for detection of bacterial contamination using microdots of immobilized whole cell-based biosensors on paper that boast nanomolar level detection of QSMs in two different food matrices, namely beef and milk. Limits of detection ranged from 1 × 10-7 M to 1 × 10-9 M with relative standard deviations (RSDs) of 1-16%. This rapid, easy, and portable test could be a useful tool for use in the field and during all stages of food manipulation, i.e., from farms to distribution, storage, sales, and preparation prior to consumption, to ensure that food is free of bacterial contamination.

Transcriptional regulatory proteins as biosensing tools.

We have developed sensing systems employing different classes of transcriptional regulatory proteins genetically and chemically modified to incorporate a fluorescent reporter molecule for detection of arsenic, hydroxylated polychlorinated biphenyls (OH-PCBs), and cyclic AMP (cAMP). These are the first examples of optical sensing systems based on transcriptional regulatory proteins.

Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field.

Alemtuzumab improves neurological functional systems in treatment-naive relapsing-remitting multiple sclerosis patients.

BACKGROUND: Individual functional system scores (FSS) of the Expanded Disability Status Scale (EDSS) play a central role in determining the overall EDSS score in patients with early-stage multiple sclerosis (MS). Alemtuzumab treatment improves preexisting disability for many patients; however, it is unknown whether improvement is specific to certain functional systems. OBJECTIVE: We assessed the effect of alemtuzumab on individual FSS of the EDSS. METHODS: CAMMS223 was a 36-month, rater-blinded, phase 2 trial; treatment-naive patients with active relapsing-remitting MS, EDSS ≤3, and symptom onset within 3 years were randomized to annual courses of alemtuzumab or subcutaneous interferon beta-1a (SC IFNB-1a) 44 µg three times weekly. RESULTS: Alemtuzumab-treated patients had improved outcomes versus SC IFNB-1a patients on most FSS at Month 36; the greatest effect occurred for sensory, pyramidal, and cerebellar FSS. Among patients who experienced 6-month sustained accumulation of disability, clinical worsening occurred most frequently in the brainstem and sensory systems. For patients with 6-month sustained reduction in preexisting disability, pyramidal and sensory systems contributed most frequently to clinical improvement. CONCLUSIONS: Alemtuzumab demonstrated a broad treatment effect in improving preexisting disability. These findings may influence treatment decisions in patients with early, active relapsing-remitting MS displaying neurological deficits. ClinicalTrials.gov Identifier NCT00050778.