UV surface disinfection in a wearable drug delivery device

The advent of recombinant DNA technology fundamentally altered the drug discovery landscape, replacing traditional small-molecule drugs with protein and peptide-based biologics. Being susceptible to degradation via the oral route, biologics require comparatively invasive injections, most commonly by intravenous infusion (IV). Significant academic and industrial efforts are underway to replace IV transport with subcutaneous delivery by wearable infusion devices. To further complement the ease-of-use and safety of disposable infusion devices, surface disinfection of the drug container can be automated. For ease of use, the desired injector is a combination device, where the drug is inside the injector as a single solution combination device. The main obstacle of the desired solution is the inability to sterilize both injector and drug in the same chamber or using the same method (Gamma for the drug and ETO for the injector). This leads to the assembly of both drug container and injector after sterilization, resulting in at least one transition area that is not sterilized. To automate the delivery of the drug to the patient, a disinfection step before the drug delivery through the injector is required on the none-sterilized interface. As an innovative solution, the autoinjector presented here is designed with a single ultraviolet light-emitting diode (UV LED) for surface disinfection of the drug container and injector interface. In order to validate microbial disinfection similar to ethanol swabbing on the injector, a bacterial 3 or 6 log reduction needed to be demonstrated. However, the small disinfection chamber surfaces within the device are incapable of holding an initial bacterial load for demonstrating the 3 or 6 log reduction, complicating the validation method, and presenting a dilemma as to how to achieve the log reduction while producing real chamber conditions. The suggested solution in this paper is to establish a correlation model between the UV irradiance distribution within the disinfection chamber and a larger external test setup, which can hold the required bacterial load and represents a worse-case test scenario. Bacterial log reduction was subsequently performed on nine different microorganisms of low to high UV-tolerance. The procedure defined herein can be adopted for other surface or chamber disinfection studies in which the inoculation space is limited.     

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Cancer progression is accompanied by extensive changes to the tumor microenvironment, which support the proliferation and dissemination of malignant cells. Extracellular matrix (ECM) remodeling by cancer and associated stromal cells utilizes a host of enzymes that act upon both proteins and carbohydrates within the ECM, causing profound compositional changes that can drive growth of the primary tumor, as well as prime distant sites for metastasis (1).Heparan sulfate proteoglycans (HSPGs) are a fundamental class of ECM constituent, comprised of pericellular and extracellular core proteins conjugated to one or more chains of the glycosaminoglycan polysaccharide heparan sulfate (HS) (2). HSPGs mediate myriad biological processes, including signaling (3), developmental patterning (4), adhesion (5), barrier formation (6, 7), endocytosis (8), and viral entry (9–11). These processes largely depend upon the HS polysaccharides adorning the core protein (12), whose heterogeneous structures allow for interaction with multiple diverse partners.Given the importance of HSPGs within the ECM, tightly coordinated biosynthesis and breakdown mechanisms act to regulate their composition (13, 14). HSPG breakdown is mediated (in part) by endo- and exo-glycosidases, which hydrolytically cleave within or at the termini of HS polysaccharide chains, respectively (14). Heparanase (HPSE) is a mammalian endo-β-D-glucuronidase (hereafter, endo-β-glucuronidase) that plays a key role in HS polysaccharide degradation (15). Initially produced as a proenzyme (proHPSE), HPSE is matured within lysosomes by proteolysis of a linker peptide that obstructs its active site. Mature HPSE remains active within lysosomes, but can also be secreted into the extracellular space, where it aids ECM remodeling during tissue development and homeostasis. In contrast to its physiological role in ECM maintenance, pathological HPSE overexpression strongly drives the growth of aggressive metastatic cancers (16): excessive HSPG degradation within the ECM directly facilitates cancer cell migration to and from the vasculature (17, 18), while growth factors and cytokines liberated upon HSPG degradation stimulate proliferation and angiogenesis (19, 20) (Fig. 1A). HPSE overexpression can also increase the formation of tumor-derived exosomes, which circulate to distal tissues to establish premetastatic niche environments primed for colonization (21–23).Open in a separate windowFig. 1.Design and development of HS-configured cyclophellitol pseudodisaccharides. (A) Biological effects of HPSE overexpression in the extracellular space. Excessive degradation of HSPG networks in basement membranes facilitates cell migration to and from the vasculature. Release of HSPG sequestered growth factors stimulates cell proliferation. (B) Preferred HPSE target sequence, comprising a GlcA residue flanked by two sulfated glucosamine residues. (C) Inhibitors and probes used in this study; atom reference positions are shown on inhibitor 2. Full structures of 7–9, including Cy5 linker, are shown in the SI Appendix. (Bottom) Principal of pseudodisaccharide HPSE selectivity via steric occlusion of exo-β-glucuronidase binding.HPSE is the sole human enzyme responsible for extracellular HS polysaccharide degradation. Accordingly, there is intense interest in its inhibition as an anticancer strategy. A plethora of small-molecule (24–27), saccharide (28–31), neoproteoglycan (32, 33), and antibody (34) based HPSE inhibitors have been reported (35), although, to date, only four compounds have progressed to clinical trials (SI Appendix, Fig. S1) (28–31). Notably, these current “best-in-class” inhibitors are all polyanionic oligo-/polysaccharide derivatives that mimic the physicochemical properties of the natural HPSE inhibitor heparin, a glycosaminoglycan polysaccharide closely related to HS. The heparin-like properties of these best-in-class inhibitors, along with their structural heterogeneity, can produce unwanted pleiotropic effects, such as anticoagulation and growth factor binding, which complicate their clinical use (36).Comparatively few small-molecule HPSE inhibitors with in vivo efficacy have been described (35). The active site of HPSE has proven challenging for small-molecule pharmacological intervention, given its extensive interaction surface evolved to bind large HS polysaccharides. Such challenging sites are often well targeted by mechanism-based covalent inhibitors, which may still react with an enzyme despite weak or transient initial binding. We previously reported that derivatives of the naturally occurring β-glucosidase inhibitor cyclophellitol yield highly active mechanism-based retaining glycosidase inhibitors and probes (37–40). Enzymatic attack by glycosidases upon the electrophilic epoxide “warhead” of cyclophellitol-derived molecules leads to alkylation of a catalytically essential nucleophile residue, resulting in covalent and irreversible inhibition (SI Appendix, Fig. S2). Tuning the functionality and stereochemistry of cyclophellitol-derived scaffolds alters target selectivity, in principle allowing inhibition of any retaining glycosidase enzyme (41).Herein, we present HS-configured pseudodisaccharides, featuring a cyclitol epoxide designed to bind selectively to the HPSE active site and react with its essential catalytic nucleophile residue. We show that these HS-configured pseudodisaccharides are nanomolar, irreversible HPSE inhibitors in vitro and markedly reduce cancer aggression in in cellulo and in vivo cancer models. Covalent irreversible inhibition remains an underexploited avenue for therapeutic exploitation. Our results demonstrate that compounds acting by this mechanism can deliver effective treatments for addressing HPSE-driven cancer aggression.     

CLDN1 Arg81His founder variant causes ichthyosis,leukocyte vacuoles,alopecia, and sclerosing cholangitis (ILVASC) syndrome in Moroccan Jews

Neonatal ichthyosis and sclerosing cholangitis syndrome (NISCH), also known as ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis (ILVASC), is an extremely rare disease of autosomal recessive inheritance, resulting from loss of function of the tight junction protein claudin-1. Its clinical presentation is highly variable, and is characterized by liver and ectodermal involvement. Although most ILVASC cases described to date were attributed to homozygous truncating variants in CLDN1, a single missense variant CLDN1 p.Arg81His, associated with isolated skin ichthyosis phenotype, has been recently reported in a family of Moroccan Jewish descent. We now describe seven patients with ILVASC, originating from four non consanguineous families of North African Jewish ancestry (including one previously reported family), harboring CLDN1 p.Arg81His variant, and broaden the phenotypic spectrum attributed to this variant to include teeth, hair, and liver/bile duct involvement, characteristic of ILVASC. Furthermore, we provide additional evidence for pathogenicity of the CLDN1 p.Arg81His variant by transmission electron microscopy of the affected skin, revealing distorted tight junction architecture, and show through haplotype analysis in the vicinity of the CLDN1 gene, that this variant represents a founder variant in Jews of Moroccan descent with an estimated carrier frequency of 1:220.     

Hormone seasonality in medical records suggests circannual endocrine circuits

Hormones control the major biological functions of stress response, growth, metabolism, and reproduction. In animals, these hormones show pronounced seasonality, with different set-points for different seasons. In humans, the seasonality of these hormones remains unclear, due to a lack of datasets large enough to discern common patterns and cover all hormones. Here, we analyze an Israeli health record on 46 million person-years, including millions of hormone blood tests. We find clear seasonal patterns: The effector hormones peak in winter−spring, whereas most of their upstream regulating pituitary hormones peak only months later, in summer. This delay of months is unexpected because known delays in the hormone circuits last hours. We explain the precise delays and amplitudes by proposing and testing a mechanism for the circannual clock: The gland masses grow with a timescale of months due to trophic effects of the hormones, generating a feedback circuit with a natural frequency of about a year that can entrain to the seasons. Thus, humans may show coordinated seasonal set-points with a winter−spring peak in the growth, stress, metabolism, and reproduction axes.

Major biological functions in mammals—growth, reproduction, metabolism, and stress adaptation—are controlled by dedicated hormonal axes. In each axis, signals from the hypothalamus cause secretion of specific pituitary hormones into the bloodstream. The pituitary hormones instruct a peripheral organ to secrete effector hormones with widespread effects on many tissues. For example, stress response is governed by the hypothalamic−pituitary−adrenal (HPA) axis: Physiological and psychological stress signals cause the hypothalamus to induce secretion of ACTH from the pituitary, which instructs the adrenal cortex to secrete cortisol. These axes act to maintain physiological set points. The set points can change to adapt to different situations, a concept known as rheostasis (1).A major reason that organisms change their set points is the seasons.Animals show seasonal changes in the pituitary and effector hormones that govern seasonality in reproduction, activity, growth, pigmentation, morphology, and migration (2). This adaptive physiology includes changes in body composition, organ size, and function. In general, hormone seasonality is thought to be a dominant regulator of physiological and behavioral traits in animals (3, 4).Animals show these changes with a circannual rhythm even when maintained in constant photoperiod and temperature conditions (3, 5, 6). They cycle without external signals, by means of an internal oscillator with a period of about, but not exactly, 1 y. The mechanism and physiological location of this circannual clock is a subject of current research. A key component is the pars tuberalis in the pituitary stalk, whose thyrotroph cells oscillate between high and low states of hormone production (7–9). This area receives input on photoperiod from melatonin signals.Whether hormones show seasonality in humans has not been studied comprehensively by tracking many hormones in a large number of participants. Each axis has been studied separately, usually with small samples. These studies suggest that thyroid hormones and cortisol show seasonal variation on the order of 10% (10, 11). The studies are limited by considerations of circadian rhythms which affect cortisol and other hormones.To study human hormone seasonality requires a large dataset with a comprehensive coverage of all hormones. Here we provide such a study using an Israeli medical record database with millions of blood tests. We address the circadian rhythm concern using the time of each test. We find coordinated seasonality with a winter/spring peak in effector hormones and a surprising antiphase between pituitary and effector hormones. We provide an explanation for this antiphase by showing that trophic effects of the hormones create a circuit in which the functional masses of the glands changes over the year and can entrain to yearly signals. The results support a winter−spring peak for human reproduction, metabolism, growth, and stress adaptation.     

Interhemispheric cortico-cortical paired associative stimulation of the prefrontal cortex jointly modulates frontal asymmetry and emotional reactivity

Background

As advances in neuroimaging further our understanding of the brain's functional connectivity, neuropsychology has moved away from a regional approach of attributing behavior to a specific region towards a network approach, attributing behavior to interconnected regions. A prime example of this is the suggested relevance of frontal asymmetry of the lateral prefrontal cortex (LPFC) in emotional processing. Yet, while neuroimaging defines relevant networks, it can only establish correlations and not causality.

In vivo assessment of aged human skin with a unilateral NMR scanner

Human skin undergoes morphological and biochemical changes as a result of chronological aging and exposure to solar ultraviolet irradiation (photoaging). Noninvasive detection of these changes may aid in the prevention and treatment of both types of aging. This article presents a noninvasive method for the evaluation of aging skin with a unilateral stray field NMR scanner. These portable and inexpensive scanners may be suitable for in‐depth skin characterization. In vivo profiles of sun‐protected and sun‐exposed skin from the forearms of female subjects of different ages (n = 9) were measured. Skin biopsies for histopathological examination were used as reference. T₂ analysis with a bi‐exponential decay model was applied and the extracted parameters were examined as markers for dermal aging. In the upper reticular dermis, a significant increase in the fraction of the slow T₂ component and in the T₂ value itself was found to correlate with chronological aging. For most subjects, there was an additional increase in the values of the slow T₂ component and the T₂ values from the sun‐exposed forearm, superimposed on that measured for the sun‐protected forearm. These results are in agreement with the decline in collagen content and the increase in free water content with aging. The results suggest that such a technique can be used as a tool for the assessment of aging, and that bi‐exponential fitting can produce sensitive fingerprint parameters for the dermal alterations that occur during aging. Copyright © 2015 John Wiley & Sons, Ltd.