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Axonal regeneration following surgical nerve repair is slow and often incomplete, resulting in poor functional recovery which sometimes contributes to lifelong disability. Currently, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery. The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for scalable production and clinical application, aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80% compared with systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.

ABSTRACTThe multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.

The goal of this paper is to investigate the effects of intonational differences across Basque varieties. We have chosen three Markina Basque contours as stimuli in a perception experiment: those that are used in declaratives, yes-no questions and verum focus sentences. Stimuli include both natural and manipulated contours. Three participant groups (with Markina Basque, another Basque variety or Spanish as L1) classified stimuli as questions or statements. The results show that Markina Basque speakers differ from the other two groups in the interpretation of the contours. Markina speakers attend mostly to the tonal contour of the last syllable to identify questions.

Abstract Diabetes is by far, the most common cause of neuropathy, inducing neurodegeneration of terminal sensory nerve fibers associated with loss of sensation, paresthesia, and persistent pain. Foretinib prevents die-back degeneration in cultured sensory and sympathetic neurons by rescuing mitochondrial activity and has been proven safe in prospective clinical trials. Here we aimed at investigating a potential neuroprotective effect of Foretinib in experimental diabetic neuropathy. A mouse model of streptozotocin induced diabetes was used that expresses yellow fluorescent protein (YFP) in peripheral nerve fibers under the thy-1 promoter. Streptozotocin-injected mice developed a stable diabetic state (blood glucose > 270 mg/dl), with a significant reduction of intraepidermal nerve fiber density by 25% at 5 weeks compared to the non-diabetic controls. When diabetic mice were treated with Foretinib, a significantly greater volume of the cutaneous nerve fibers (67.3%) in the plantar skin was preserved compared to vehicle treated (37.8%) and non-treated (44.9%) diabetic mice while proximal nerve fiber morphology was not affected. Our results indicate a neuroprotective effect of Foretinib on cutaneous nerve fibers in experimental diabetic neuropathy. As Foretinib treated mice showed greater weight loss compared to vehicle treated controls, future studies may define more sustainable treatment regimen and thereby may allow patients to take advantage of this neuroprotective drug in chronic neurodegenerative diseases like diabetic neuropathy.

Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections. Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain. To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats. The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence. The optical clearing was compatible with conventional laboratory techniques, including retrograde labeling studies, and computational image segmentation, providing fast and precise cell quantitation. Further, optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies, tracing of intramuscular nerve branches or mapping of neurovascular networks. Given their wide-ranging applicability, rapid processing times, and low costs, tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies. All procedures were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee (49871/9) on November 9, 2019.

In this study we examine the effects of word boundaries on the lenition of intervocalic voiceless plosives in Catalan in order to test the role of phonological contrastiveness in phonetic processes. Here we test the hypothesis that word-final intervocalic voiceless plosives (VC#V) will show greater lenition than word-internal and word-initial intervocalic tokens (VCV, V#CV), since in word-final position the contrast between /ptk/ and /bdg/ is neutralized. Lenition should be manifested acoustically as greater intensity, shorter duration and greater voicing. We find weaker support for the hypothesis than in a parallel study on Basque, suggesting the existence of phonological differences between the two languages. On the other hand, we find a strong effect of style on intervocalic lenition, with conversational speech promoting more lenited consonants. Intervocalic stop lenition in Catalan does not appear to be driven by temporal reduction.

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies.Graphical abstract: Cartoon of explant skin culture. Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

Abstract The current study is aimed at preparing light-driven novel functional AgNPs- bio-hydrogel and evaluating anticancer potency against human melanoma cells. With an average size of 16–18 nm, the hydrogel nano-silver particle composite (AgNPs@C_MA_O) was synthesized using a soft white LED approach and analyzed by UV–Vis, DLS, FTIR, X-ray, SEM–EDX and TEM techniques. The anticancer activity of the obtained novel functionalized AgNPs@C_MA_O was tested in-vitro in the A375 melanoma cell line. Dose–response analysis showed that AgNPs at 0.01 mg/mL and 0.005 mg/mL doses reduced the viability of A375 cells by 50% at 24 and 48-h time-points, respectively. A375 cells treated with AgNPs@C_MA_O for 24 h at IC50 displayed abnormal morphology such as detachment edges and feet, shrinkage, membrane damage, and the loss of contact with adjacent cells. Our work is the first study showing that non-ionizing radiation mediated biofunctionalized AgNPs have an anti-tumoral effect at such a low concentration of 0.01 mg/mL. Our approach of using harmless wLED increased synergy between soft biopolymer compounds and AgNPs, and enhanced anticancer efficiency of the AgNPs@C_MA_O biohydrogel. Ultimately, the AgNPs accessed through the use of the wLED approach in colloidal syntheses can open new applications and combinatorial advanced cancer treatments and diagnostics.

The notion of marginal contrasts and other gradient relations challenges the classification of phones as either contrastive phonemes or allophones of the same phoneme. The existence of “fuzzy” or “intermediate” contrasts has implications for language acquisition and sound change. In this research, we examine production and perception of two marginal contrasts [ɑ-ɔ] (“cot-caught”), where two original phonemes are undergoing a merger, and [ʌi-aɪ] (“writer-rider”), where a single original phoneme has arguably split into two contrastive sounds, albeit in a limited manner. Participants born and raised in Illinois were asked to provide recordings of cot-caught and writer-rider pairs embedded in sentences, followed by the target word in isolation. They then completed ABX and two-alternative forced choice two-alternative forced choice (2FC) perception tasks with stimuli produced by two native speakers from the Chicagoland area. Results showed that the [ʌi-aɪ] contrast, which has been defined as marginal in other work, is actually currently more phonetically and phonologically stable than [ɑ-ɔ] for the group of speakers that we have tested, with a more robust link between production and perception. The cot-caught merger appears to have progressed further, compared to what had previously been documented in the region. Our results and analysis suggest different sound change trajectories for phonological mergers, regarding the coupling of production and perception, as compared with phonemic splits.

It is not yet clear how ubiquitously-expressed proteins can cause the selective degeneration of particular populations of neurons, such as in spinocerebellar ataxia type 17, SCA17, which results from a CAG trinucleotide repeat expansion in the ubiquitously expressed transcription factor TBP. Here, the authors show that mutant TBP suppresses the cerebellum-enriched transcription of Inpp5a and link altered levels of INPP5A to the selective degeneration of cerebellar neurons.

Euskaraz hitz-bukaerako herskariek oso maiztasun txikia dute hiztegian, baina oso maiztasun handia solasean; /-t/ eta /-k/ kontsonanteak maiztasun handiko atzizki batzuetan aurkitzen baitira. Lan honetan hitz-bukaerako herskarien gauzatze fonetikoa aztertzen dugu, eta bereziki bi hitzen arteko kontsonante taldeetan jartzen dugu arreta. Datuak elkarrizketa libretik atera ditugu. Intentsitate neurketak erakusten du talde horietan kontsonanteak oso ahulak direla gehienetan. Espektrogramak miatuz baieztatzen dugu /-k/ eta /-t/ kontsonanteak beste kontsonante baten aurrean gehiengoetan galtzen edo hurbilkari ahostun gisa ahoskatzen direla. Bestalde, ahoskabetze gertakariak (adib. /k#b/ --> [p]) ez dira batere normalak gure corpusean.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure–function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure–function relationship of terminal galactose to complement activation in mAb therapeutics.

ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.

Inherited cardiomyopathies are among the major causes of heart failure and associated with significant mortality and morbidity. Currently, over 70 genes have been linked to the etiology of various forms of cardiomyopathy, some of which are X-linked. Due to the lack of appropriate cell and animal models, it has been difficult to model these X-linked cardiomyopathies. With the advancement of induced pluripotent stem cell (iPSC) technology, the ability to generate iPSC lines from patients with X-linked cardiomyopathy has facilitated in vitro modelling and drug testing for the condition. Nonetheless, due to the mosaicism of the X-chromosome inactivation, disease phenotypes of X-linked cardiomyopathy in heterozygous females are also usually more heterogeneous, with a broad spectrum of presentation. Recent advancements in iPSC procedures have enabled the isolation of cells with different lyonisation to generate isogenic disease and control cell lines. In this review, we will summarise the current strategies and examples of using an iPSC-based model to study different types of X-linked cardiomyopathy. The potential application of isogenic iPSC lines derived from a female patient with heterozygous Danon disease and drug screening will be demonstrated by our preliminary data. The limitations of an iPSC-derived cardiomyocyte-based platform will also be addressed.

Purpose: The cornea is the window through which we see the world and is one of the most densely innervated structures in the body. Besides providing protective sensory input, corneal nerves have been postulated to stimulate limbal stem cells (LSCs), hence governing corneal epithelial maintenance and recovery. Loss of corneal innervation, through injury, diabetes, tumors, infections, and even improper contact lens use, leads to neurotrophic keratopathy (NK), a degenerative corneal disease that is characterized by corneal epithelial breakdown, scarring, and permanent vision loss1. The only non-invasive treatment option for NK is the human recombinant nerve growth factor (rhNGF), but the short half-life of exogenous neurotrophins-based therapies make this therapeutic approach less effective2. Development of the small molecule ligands for neurotrophins receptors that have better pharmacokinetics and plasma stability showed promising results in the treatment of several neurodegenerative conditions in the recent years3. In this study, we were prompted to investigate the molecular mechanism of NK and the role of NGF receptors, TrkA and p75NTR, in corneal healing. We hypothesized that TrkA inhibition would delay corneal wound healing and p75NTR inhibition accelerates corneal healing. This knowledge will lay the basis for a new non-invasive approach for NK. Methods: For this experiment, we took advantage of commercially available Ntrk1 mutant mice, which allow for pharmacological inhibition of TrkA receptor with an inhibitor known as not mammalian kinase inhibitor (1-NM-PP1)4. Ntrk1 mice (n=20) were divided into three groups, which received saline injection as a control. In one experimental group animals were received TrkA inhibitor and the other group received both TrkA and p75 inhibitor for 5 days. On day six we removed the corneal epithelium with a 0.5 mm rotating brush. To measure epithelial healing, we performed digital imaging of fluorescein staining daily for four days after injury. We then harvested the corneas for immunofluorescent and biochemical analyses. Results: Our results show a significant delay in corneal epithelial healing following TrkA inhibition and acceleration in corneal healing after p75NTR inhibition. Conclusion: A selective TrkA agonist or p75NTR inhibitors could be a new therapeutic approach for NK.

Polycystic kidney disease (PKD) is the most common inherited cause of kidney failure and currently has limited treatment options. Increasing water intake reduces renal cyst growth in the pck rat (a genetic ortholog of autosomal recessive PKD) but it is not clear if this beneficial effect is present in other models of PKD. In this study, we tested the hypothesis that high water intake (HWI) reduces the progression of cystic renal disease in Lewis polycystic kidney (LPK) rats (a genetic ortholog of human nephronophthisis-9). Groups of female and male LPK (n = 8-10 per group) and Lewis (n = 4 per group) rats received water ad libitum supplemented with or without 5% glucose [to simulate HWI or normal water intake (NWI) respectively] from postnatal weeks 3 to 16. Water intake increased ~1.3-fold in the LPK+HWI group compared to LPK+NWI rats between weeks 3 to 10 but the differences were not significant at later timepoints. In LPK rats, HWI reduced the increases in the kidney to body weight ratio by 54% at week 10 and by 42% at week 16 compared to NWI (both p

Maintaining hydration sufficient to reduce levels of arginine vasopressin has been hypothesised to slow kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD). The semi-quantitative beverage frequency questionnaire (BFQ) was designed to measure usual fluid intake over the past month. The aim of this study was to assess the validity and reliability of the BFQ compared with the 24-h urine biomarkers. Participants with ADPKD (18–67 years; estimated glomerular filtration rate (eGFR) ≥ 30 mL/min1.73 m2) completed the BFQ. Serum creatinine, eGFR, 24-h urine volume, and osmolality were measured. Pearson correlation coefficients, paired t test, and Bland–Altman plots were used to evaluate agreement between the methods. A subset repeated the BFQ to assess reliability. A total of 121 participants (54% male, 43 ± 11 years; mean ± SD) completed the BFQ and at least one 24-h urine collection. The correlation between the BFQ and the 24-h urine volume was moderate (r = 0.580) and weaker with the 24-h urine osmolality (r = −0.276). The Bland–Altman plots revealed good agreement between the BFQ and the 24-h urine volume with no obvious bias; however, the limits of agreement were wide (−1517–1943 mL). The BFQ1 and BFQ2 were strongly correlated (r = 0.799, p < 0.001) and were not significantly different (p = 0.598). The BFQ is a valid and reliable tool to assess the usual fluid intake of the ADPKD population.

We hypothesized that the brain-protective effect of hyperbaric oxygen (HBO) preconditioning in a transient global cerebral ischemia rat model is mediated by the inhibition of early apoptosis.One hundred ten male Sprague–Dawley (SD) rats (300–350 g body weight) were allocated to the sham group and three other groups with 10 min of four-vessel occlusion, untreated or preconditioned with either 3 or 5 hyperbaric oxygenations. HBO preconditioning improved neurobehavioral scores and reduced mortality, decreased ischemic cell change, reduced the number of early apoptotic cells and hampered a conversion of early to late apoptotic alterations. HBO preconditioning reduced the immunoreactivity of phosphorylated p38 in vulnerable neurons and increased the expression of brain derived neurotrophic factor (BDNF) in early stage post-ischemia. However, preconditioning with 3 HBO treatments proved less beneficial than with 5 HBO treatments.We conclude that HBO preconditioning may be neuroprotective by reducing early apoptosis and inhibition of the conversion of early to late apoptosis, possibly through an increase in brain BDNF level and the suppression of p38 activation.