Penetration Assay Research Articles

Nanoemulsions containing Garcinia mangostana L. pericarp extract for topical applications: Development, characterization, and in vitro percutaneous penetration assay.

A highly stable oil-in-water nanoemulsion for topical applications, containing mangostins extracted from the pericarp of mangosteen (Garcinia mangostana L.), is a promising strategy to protect mangostins as well as to improve penetration of these important antioxidants through the skins. Nanoemulsions consisted of virgin coconut oil as the oil phase, Tween-80 and Span-80 as surfactants, and xanthan gum as the thickening agent, were prepared using the high-energy and low-energy emulsification methods. The nanoemulsions that were stable up to 28 days had oil droplet diameter of 220 nm to 353 nm and zeta potential of -46.9 mV to -63.7 mV. The accelerated stability test showed that the most stable nanoemulsions were those prepared using the low-energy emulsification method with an estimated shelf life of eleven months, composed of 11% oil phase, 17% surfactant, and 72% aqueous phase. The in vitro percutaneous penetration test for the nanoemulsion with added xanthan gum provided high cumulative skin penetration of mangostins of up to 114 μg/cm2. The results of this study indicate that virgin coconut oil-based nanoemulsions containing mangostins, prepared using the low-energy emulsification method, stabilized by xanthan gum and mixed at 40°C can prospectively be used for topical applications.

MCM3AP-AS1 regulates proliferation, apoptosis, migration, and invasion of breast cancer cells via binding with ZFP36.

BackgroundPrevious studies suggest that long noncoding RNA (lncRNA) maintenance complex component 3 associated protein (MCM3AP) antisense RNA 1 (MCM3AP-AS1) has a wide range of functions in several cancers. However, its expression and functions in breast cancer are unclear.MethodsReverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of MCM3AP-AS1. MTT, colony formation and anchorage-independent growth assay were used to examine the effect of MCM3AP-AS1 on growth of breast cancer cells. TUNEL and flow cytometry assay were applied to investigate the role of MCM3AP-AS1 on cell apoptosis. Wound heading and transwell matric penetration assay were used to detect the role of MCM3AP-AS1 on cell motility. RNA pull-down and RIP assay were used to examine the interaction of MCM3AP-AS1 and ZFP36.ResultsWe found that lncRNA MCM3AP-AS1 was significantly upregulated in breast cancer, which correlated with patients’ clinicopathological characteristics. Downregulation of MCM3AP-AS1 substantially inhibited the proliferation, apoptosis, migration, and invasion of breast cancer cells. Further analysis clarified that MCM3AP-AS1 binds with the RNA-binding protein ZFP36 ring finger protein (ZFP36) to regulate the levels of cyclin D1 (CCND1), c-myc (MYC), and matrix metalloproteinase 1 (MMP1).ConclusionsOur findings show lncRNA MCM3AP-AS1 might be a prognostic factor and a promising therapeutic target for breast cancer therapy.

Pterostilbene Attenuates Particulate Matter-Induced Oxidative Stress, Inflammation and Aging in Keratinocytes.

Particulate matter (PM) is the main indicator of air pollutants, and it may increase the level of reactive oxygen species (ROS) in keratinocytes, leading to skin inflammation, aging, and decreased moisturizing ability. Pterostilbene (PTS) is a dimethylated analog of resveratrol that has antioxidant effects. However, the molecular mechanisms of PTS in preventing PM-induced keratinocyte inflammation and aging have not been investigated yet. Therefore, we used PM-induced human keratinocytes to investigate the protective mechanisms of PTS. The results showed that 20 μM PTS had no toxicity to HaCaT keratinocytes and significantly reduced PM-induced intracellular ROS production. In addition, nuclear translocation of the aryl hydrocarbon receptor (AHR) was inhibited by PTS, leading to reduced expression of its downstream CYP1A1. PTS further inhibited PM-induced MAPKs, inflammation (COX-2), and aging (MMP-9) protein cascades, and rescued moisturizing (AQP-3) protein expression. We analyzed the PTS content in cells at different time points and compared the concentration required for PTS to inhibit the target proteins. Finally, we used the skin penetration assay to show that the PTS essence mainly exists in the epidermal layer and did not enter the system circulation. In conclusion, PTS could protect HaCaT keratinocytes from PM-induced damage and has the potential to become a cosmetic ingredient.

Susceptibility of the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae), to four species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from Mizoram, North-Eastern India

BackgroundOutbreak of the fall armyworm Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) occurred in Mizoram, North-Eastern India. The infestation spread in the entire state covering a total area of around 2840 hectares of maize cultivated land. Entomopathogenic nematodes (EPNs) represent potential candidates for the biological control of S. frugiperda. In the study, the susceptibility of the pest against 4 locally isolated EPN species Heterorhabditis indica, H. baujardi, Steinernema sangi and S. surkhetense was evaluated.ResultsThe results indicated that all the isolated EPN species showed a high rate of larvicidal and pupicidal activities against the pest. Mortality between 43.75–100.00 and 25.00–100.00% of 3rd and 5th larval instars, respectively (at concentrations 10–800 IJs/larva), and 37.50–68.75% mortality of pupae (at concentrations 200–1600 IJs /pupa) were found after exposure to the EPN species. The mortality rate of the pest showed significant variations with life stages of the host insect, nematode concentrations and incubation time. Based on the median lethal concentration (LC50), H. indica was the most pathogenic species, followed by S. sangi, H. baujardi and S. surkhetense. The LC50 values of H. indica at 72 h post-incubation were 20.26 and 62.07 IJs/larva for the 3rd and 5th larval instars, respectively, and 913.34 IJs/pupa. The penetration assay showed that H. indica had the highest penetration rate into the hosts, 27.24, 21.30 and 20.00% in the 3rd, 5th larval instars and pupae, respectively. Furthermore, all the EPN isolates were capable of successful multiplication inside the cadaver of S. frugiperda that showed significant differences with the EPN isolates and life stages of the pest. Among the isolates, H. indica showed the highest multiplication rates, 17,692.25 ± 2103.59, 8345.63 ± 785.34 and 79,146.38 ± 5943.73 IJs per 3rd instar larva, 5th instar larva and pupa, respectively.ConclusionsThe study revealed that the 4 species of EPNs showed a high potency against S. frugiperda, thereby having the potential to be developed as a biological control agent against the pest. Moreover, the isolated EPN species could potentially serve as alternatives for chemical insecticides and could further be incorporated into the Integrated Pest Management (IPM).

Catalase-loaded polymersomes as a promising safe ingredient to active photoprotection

The topical application of enzymes for UV-induced damage repair, such as catalase, represents an interesting strategy for "active" photoprotection. However, catalase, a large and hydrophilic molecule, needs to penetrate the skin to present activity and, in this sense, nanocarriers can provide an aid for barrier crossing. Among the nanocarriers, polymersomes (Ps) obtained by the self-aggregation of amphiphilic block copolymers represent an alternative for topical enzyme delivery. Here, catalase-loaded Ps of Pluronic® L121, an amphiphilic triblock copolymer, were prepared by direct dissolution and in vitro evaluated regarding safety and efficacy. The Ps presented a suitable photo/cytotoxicity profile regarding the sun-damaged epidermis. Pre-clinical safety tests indicated that the HET-CAM assay was more appropriate than cytotoxicity assays to predict the irritant potential of the Ps. Based on cutaneous permeation assays associated with laser scanning confocal fluorescence microscopy, the Ps increased ten times the catalase percentage of skin permeation. Additionally, in vitro assays indicated that catalase-loaded Ps were able to eliminate UV-induced lipid peroxidation in deeper layers of the skin (viable epidermis + dermis) in comparison to free catalase. Penetration assays in 3D cell culture and efficacy assays confirmed the preferential delivery of catalase in the deeper layers of the skin owing to the permeation enhancer effect of Pluronic L121 and/or the membrane translocation capacity of the Ps. In conclusion, our findings indicate catalase-loaded Ps as an innovative nanomaterial for active sunscreens.

Allicin-loaded chitosan/polyvinyl alcohol scaffolds as a potential wound dressing material to treat diabetic wounds: An in vitro and in vivo study

In the current study an Allicin delivering polymeric wound dressing was formulated using thermally induced phase separation method. Allicin was loaded into Chitosan/Polyvinyl Alcohol solution at weight ratios 3 %,5 %, and 10 % and then freeze-dried. The fabricated membrane's physiochemical and biological properties were assessed using scanning electron microscopy, cell viability assay, swelling capacity, mechanical strength, surface wettability, water vapor permeability, drug release profile, in vitro degradation, hemocompatibility, porosity measurement, microbial penetration assay, Anti-oxidant assay, and protein adsorption assay. According to cell MTT assay results, Chitosan/Polyvinyl Alcohol incorporated with 5 % Allicin had the highest cell viability compared with other formulations; therefore, this formulation was chosen for treating rat model of diabetic wounds. The results showed that the rats treated with Allicin loaded dressings exhibited 93.15 ± 6.38 % of wound closure after 14 days. In addition, this group showed 47.11 ± 3.67 μm of epithelial thickness and 72.31 ± 4.28% of collagen deposition at the end of 14th post-wounding. These values were significantly greater than that of Allicin-free dressings and control group, p value < 0.05. These preliminary results suggest potential applicability of Allicin loaded Chitosan/Polyvinyl Alcohol dressing in treating diabetic wounds in clinic.

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships.

The efficiency with which polycationic peptides penetrate the cytosol depends on the number and overall patterning of arginine residues. While general trends and unusually penetrant patterns of arginine residues have been discovered, prior work has not effectively leveraged high-throughput screens to measure cytosolic penetration rather than total cell uptake. In this work, we adapted the chloroalkane penetration assay, which exclusively measures cytosolic penetration, to screen peptide libraries in a high-throughput, quantitative, and automation-ready manner. We demonstrate the usefulness of the screening platform by efficiently exploring how the number, patterning, and stereochemistry of arginine residues affect the cytosolic penetration of a model 10-residue peptide.

Effect of chronically electric foot shock stress on spatial memory and hippocampal blood brain barrier permeability

Maintaining blood-brain barrier (BBB) contributes critically to preserving normal brain functions. According to the available evidence, intense or chronic exposure to stress would potentially affect different brain structures, such as the hippocampus, negatively. The purpose of this study was to define the relationship between the BBB permeability of the hippocampus and the performance of spatial learning and memory under chronically electric foot shock stress. Sixteen rats were divided into the control and stress groups equally. Animals in the stress group were exposed to foot shock (1 mA, 1 Hz) for 10-s duration every 60 s (1 h/day) for 10 consecutive days. The anxiety-related behavior, spatial learning, and memory were assessed by an Open Field (OF) and the Morris Water Maze (MWM) respectively. The hippocampal BBB permeability was determined by Evans blue penetration assay. Our results demonstrated that the stress model not only increased locomotor activities in the OF test but reduced spatial learning and memory in MWM. Moreover, these effects coincided with a significant increase in hippocampal BBB permeability. In sum, the stress model can be used in future studies focusing on the relationship between stress and BBB permeability of the hippocampus.

174 Knock-down of SDR9C7 impairs epidermal barrier function

The Mendelian Disorders of Cornification consist of a highly heterogeneous group of disorders, and the majority of non-syndromic cases belong to the family of autosomal recessive congenital ichthyosis (ARCI). Mutations in SDR9C7 have been associated with ACRI, and clinical manifestations include mild to moderately dry, scaly skin with or without hyperkeratosis, palmoplantar keratoderma, and erythroderma. SDR9C7, with short-chain dehydrogenase/reductase activity, is known as NAD- or NADP-dependent oxidoreductase and has been shown to be involved in the final step of epidermal lipid barrier formation by covalent binding of acylCer to the cornified envelope. Here, we present the clinical and molecular description of 19 ARCI patients in five consanguineous families with SDR9C7 mutations. We also downregulated the expression of SDR9C7 in keratinocytes by siRNA technique in 3D organotypic skin constructs. Our results demonstrated morphological and histological abnormalities in these constructs ex vivo, similar to those observed in ichthyosis patients. Moreover, the results from keratinocyte migration and epidermal dye penetration assays provided evidence for the role of SDR9C7 in the disease pathomechanism. Collectively, our results indicate that SDR9C7 deficiency by itself is sufficient to disrupt epidermal barrier function leading to ichthyotic phenotype.

HaloTag Forms an Intramolecular Disulfide.

HaloTag is a modified haloalkane dehalogenase used for many applications in chemical biology including protein purification, cell-based imaging, and cytosolic penetration assays. While working with purified, recombinant HaloTag protein, we discovered that HaloTag forms an internal disulfide bond under oxidizing conditions. In this work, we describe this internal disulfide formation and the conditions under which it occurs, and we identify the relevant cysteine residues. Further, we develop a mutant version of HaloTag, HaloTag8, that maintains activity while avoiding internal disulfide formation altogether. While there is no evidence that HaloTag is prone to disulfide formation in intracellular environments, researchers using recombinant HaloTag, HaloTag expressed on the cell surface, or HaloTag in the extracellular space might consider using HaloTag8 to avoid intramolecular disulfide formation.

Topical tacrolimus nanocapsules eye drops for therapeutic effect enhancement in both anterior and posterior ocular inflammation models

Tacrolimus has shown efficacy in eye inflammatory diseases. However, due to the drug lability, its formulation into a stable ophthalmic product remains a challenge. Tacrolimus-loaded nanocapsules (NCs) were designed for ocular instillation. Further, the stability and effects of the formulation were analyzed under different experimental conditions. Physicochemical characterization of the NCs revealed suitable homogeneous size and high encapsulation efficiency. Moreover, the lyophilized formulation was stable at ICH long term and accelerated storage conditions, for at least 18 and 3 months, respectively. The tacrolimus NCs did not elicit any eye irritation in rabbits after single- and multiple-dose applications. Additionally, ex vivo penetration assays on isolated porcine cornea and pharmacokinetics analyses in various rabbit eye compartments demonstrated the superiority of the NCs in retention and permeation into the anterior chamber of the eye compared to the free drug dissolved in oil. Moreover, multiple dose ocular instillation of the NCs in rats allowed high tacrolimus levels in the eye with very low plasma concentrations. Finally, the developed delivery system achieved a significant decrease in four typical inflammatory markers in a murine model of keratitis, an anterior chamber inflammation. Furthermore, these NCs, applied as eye drops, displayed clinical and histological efficacy in the mainly posterior chamber inflammation model of murine, experimental auto-immune uveitis.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of β-lactam antibiotics and β-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While β-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

In Vitro Analysis of the Antioxidant and Antiviral Activity of Embelin against Herpes Simplex Virus-1.

Herpes simplex virus-1 (HSV-1) causes a wide range of infections from mild to life-threatening in the human population. There are effective treatments for HSV-1 infections that are limited due HSV-1 latency and development of resistance to current therapeutics. The goal of this study was to investigate the antioxidant and antiviral effects of embelin on HSV-1 in cultured Vero cells. Oxidative stress was verified by an extensive production of a reactive oxygen species (ROS) H2O2. Vero cells were infected with a recombinant strain of HSV-1 and antiviral assays, time course attachment, penetration, and post penetration assays, confocal microscopy, qPCR, and antioxidant assays were conducted. Our results lead to the conclusion that embelin is noncytotoxic at concentrations tested ranging from 20 to 70 µM. Treatment of HSV-1 virions with embelin resulted in 98.7–100% inhibition and affected the early stage of HSV-1 infection of Vero cells, by inhibiting the attachment and penetration of HSV-1 virions to host cells. Treatment of virions with concentrations of embelin ranging from 35 to 60 µM significantly reduced the production of H2O2. In conclusion, embelin reduces oxidative damage caused by HSV-1 infection and is an effective antiviral to reduce the infection of HSV-1 in cultured Vero cells. Further studies are needed to explore the possibility of embelin as a medicinal agent.

Amphotericin B-loaded deformable lipid vesicles for topical treatment of cutaneous leishmaniasis skin lesions.

Cutaneous leishmaniasis (CL), the most common clinical form of human leishmaniasis, is a non-fatal chronic and disabling disease characterized by erythema and nodular or ulcerative skin lesions that may cause permanent scars and disfigurement. Topical drug delivery represents a simple and efficacious approach to treat CL skin lesions. The association of drugs with nanocarrier systems enhances their permeation properties and increases the drug amount available in the dermis. Here, a deformable lipid vesicle (DLV) was optimized for the topical administration of Amphotericin B (AmB), with the aim of studying and understanding the advantages of this type of delivery system in the transport of a drug through the skin layers. AmB-DVL were characterized in terms of incorporation parameters, stability, and elasticity, and evaluated in vitro for their permeation properties, cytotoxicity, and anti-leishmanial activity. The AmB-DVL exhibited a translucent fluid gel-like aspect and a yellow color, a mean size of 132nm (PdI≤0.1), zeta potential values around zero (mV), and an AmB incorporation efficiency of 95%. Permeation and penetration assays suggest that AmB-DLV are suitable for topical administration since AmB was detected in the epidermal and dermal skin layers. AmB-DVL was able to reduce promastigote viability in a dose-dependent manner, as well as the number of intracellular amastigotes in THP-1 macrophages. Selectivity index (SI) value for AmB-DLV was considerably higher than that observed for free AmB. Results suggest that DLV may represent an attractive vehicle for dermal delivery of AmB and a new low-cost and safe therapeutic option in CL treatment.

Abstract PO051: Enhancing NK cell penetration of the tumor stroma using gene modification and nanomedicine

Abstract Adoptive cell therapy has shown great success in treating liquid tumors. While T cells have been most commonly used for these indications, NK cells are emerging as a promising immune effector cell against solid tumors. Solid tumors present a more heterogeneous population of tumor antigens than liquid tumors, and NK cells, unlike T cells, can target multiple antigens to decrease the likelihood of antigen escape. NK cells can also be transplanted allogeneically with a lower risk of inducing graft-versus-host disease than T cells, which makes off-the-shelf NK cell therapy both simpler than T cell therapy and available to leukopenic patients. However, barriers to treating solid tumors with adoptive cell therapy remain, including the physical barrier of the dense stroma, which blocks immune cell infiltration. This project aims to develop an adoptive cell therapy to overcome this barrier. We tested the ability of cord blood-derived NK cells to kill mesothelioma tumor cell lines H226 and H2452 in the presence of heparanase, a heparan sulfate proteoglycan endoglucuronidase that both degrades the tumor stroma and stimulates NK cell invasion. We also conjugated this heparanase to FDA-approved Prussian blue nanoparticles to determine whether these nanoparticles might increase heparanase accumulation in the tumor and protect it from degradation. We used cytotoxicity assays, transwell invasion assays, and spheroid coculture assays to determine whether heparanase or hep-PBNPs aid NK cell cytotoxicity, invasion, and penetration. Surprisingly, our results indicate that PBNPs themselves may have heparanase activity. However, our invasion and penetration assays must be repeated to fully elucidate the effect of PBNPs and hep-PBNPs on NK cell invasion. Citation Format: Nicole F. Bonan, Eric Yvon, Rohan Fernandes. Enhancing NK cell penetration of the tumor stroma using gene modification and nanomedicine [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO051.

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

BackgroundHepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase‐dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase‐independent apoptosis has not been investigated.ObjectivesTo evaluate the effects of HBs exposure on sperm dysfunction by activating caspase‐independent apoptosis.Materials and methodsSpermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 μg/mL for 3 h. Flow cytometry, qRT‐PCR, immunofluorescence assay, ELISA, and zona‐free hamster oocyte penetration assays were performed.ResultsWith increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2‐positive cells declined and that of both Bax‐positive cells and Apaf‐1–positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf‐1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant.ConclusionHBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G–mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV‐infected patients.

An Insight into Novel Sperm Cell Proteins as Bio-markers for Male Infertility: A Review.

Male infertility is rising nowadays and accounts for a major part of infertility cases worldwide. Novel tests are being developed for better detection and management of male infertility. Though there are many tests available for diagnosing male infertility like acrosome reaction rate, hemizona assay, in vivo or in vitro sperm penetration assay, sperm DNA damage tests, however, a semen analysis is the most commonly used initial test for male infertility. It is usually associated with failure to detect the cause in many cases, as seminal composition gets affected by a number of factors and can give false reports. Furthermore, it does not give any information about defects in capacitation, sperm-zona pellucida interaction, and sperm's ability to fertilize oocytes. This results in failure of detection and delayed management of male infertility. Hence, the present review was conducted to identify various sperm proteins that play a significant role in spermatogenesis, sperm motility, sperm-zona pellucida interaction, and fertilization. These proteins can be used in the future as markers of male infertility and will aid in better detection and management of male infertility. Methodology: Search for literature was made from 1970 to 2020 from various databases like PUBMED, SCOPUS, Google Scholar on sperm proteins and their role in male fertility using keywords: "sperm protein as bio-markers", "novel sperm proteins as markers of infertility", "Sperm proteins essential for capacitation, sperm motility and oocyte fertilization". Inclusion criteria: All full-length research articles, systematic reviews, meta-analyses, or abstracts on sperm proteins and male infertility published in the English language in peer-reviewed journals were considered.

Knockdown of SDR9C7 Impairs Epidermal Barrier Function

The Mendelian disorders of cornification consist of a highly heterogeneous group of diseases, and the majority of nonsyndromic cases belong to the family of autosomal recessive congenital ichthyosis. Mutations in SDR9C7 have been associated with autosomal recessive congenital ichthyosis, and clinical manifestations include mild to moderately dry, scaly skin with or without hyperkeratosis, palmoplantar keratoderma, and erythroderma. SDR9C7, with short-chain dehydrogenase and/or reductase activity, is known as nicotinamide adenine dinucleotide‒ or nicotinamide adenine dinucleotide phosphate‒dependent oxidoreductase and has been shown to be involved in the final step of epidermal lipid barrier formation by covalent binding of acylceramide to the cornified envelope. In this study, we present the clinical and molecular description of 19 patients with autosomal recessive congenital ichthyosis in five consanguineous families with SDR9C7 mutations. We also downregulated the expression of SDR9C7 in keratinocytes using the small interfering RNA technique in three-dimensional organotypic skin constructs. Our results demonstrated morphological and histological abnormalities in these constructs exvivo, similar to those observed in patients with ichthyosis. Moreover, the results from keratinocyte migration and epidermal dye penetration assays provided evidence for the role of SDR9C7 in the disease pathomechanism. Collectively, our results indicate that SDR9C7 deficiency by itself is sufficient to disrupt epidermal barrier function leading to ichthyotic phenotype.

Myeloperoxidase inhibitor AZD5904 enhances human sperm function in vitro.

Does AZD5904, a myeloperoxidase inhibitor (MPOi), have any effect on human sperm function in vitro? AZD5904 improves sperm function in an in vitro model of oxidative stress (OS) and potentially offers a novel treatment approach for male infertility. Male infertility is an underlying or contributory cause in half of all couples experiencing difficulties conceiving, yet there is currently no effective treatment or cure. OS is a common pathology in a significant proportion of infertile men. It can negatively affect sperm motility and the ability to fertilize a mature oocyte, as well as DNA integrity, and therefore represents an attractive target for therapeutic intervention. This study included population-based samples from men (23-50 years) attending Ninewells Assisted Conception Unit, Dundee for diagnostic semen analysis, July 2017-September 2018. Semen samples (n = 47) from 45 patients were used. Neutrophils activated using zymosan were incubated with prepared human spermatozoa for 2 h (T2) and 24 h (T24) to create an in vitro model of OS. Parallel samples were co-incubated with AZD5904, an MPOi, to examine its effects. Sperm motility was assessed by computer-assisted sperm analysis at T2 and T24. Functional motility was assessed by sperm penetration assay. Statistical analysis was performed using GraphPad Prism. There was no significant difference in total or progressive sperm motility between any treatment and control groups at T2 or T24. Nonetheless, significant positive effects on sperm function were observed with AZD5904, with 16/45 (35.6%) samples (with both normal and abnormal baseline semen analysis characteristics) displaying a ≥20% increase in sperm penetrated through viscous media (P < 0.003). This was an in vitro study. Treatment with AZD5904 resulted in significant increased sperm penetration in one of three samples treated, which is likely to represent improvement in sperm function required for fertilization. We are now planning a clinical trial to validate these results and hope that this could represent a new treatment for male infertility. AZD5904 was shared through the AstraZeneca Open Innovation program. The study was funded by AstraZeneca and sponsored by the University of Dundee. Additional funding was provided by Chief Scientist Office/NHS Research Scotland (S.J.M.d.S.). A.W. and H.J.S. are both full time employees of AstraZeneca. A.W. and H.J.S. are inventors on a patent filed by AstraZeneca titled MPOi for use in medicine which includes MPOi for use in the treatment of male infertility (WO 2019/016074 Al). S.J.M.d.S. is Associate Editor of Human Reproduction and Editorial Board member of Reproduction & Fertility. C.L.R.B. is Editor of RBMO and has received lecturing fees from Merck and Ferring and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B. was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016). C.L.R.B. has a patent WO2013054111 A1 issued. The other authors declare no conflict of interest. N/A.

Hydrogen Peroxide Has Adverse Effects on Human Sperm Quality Parameters, Induces Apoptosis, and Reduces Survival.

Background:One of the causes of male fertility disorders is the exposure of oxidative stress on the human sperm. Understanding the mechanism of disturbance is important to develop a better treatment for infertile or subfertile patients.Aims:The aim of this study was to analyze the effects of hydrogen peroxide (H2O2) on human sperm quality parameters and cell survival.Settings and Design:This study used an experimental design.Materials and Methods:Sperm cells from 15 donors were washed in a Percoll gradient and dissolved in Biggers, Whitter, and Whittingham medium. Cells were incubated with H2O2 at various concentrations from 0 to 250 μM for 2 h. Sperm viability was examined by eosin assay, sperm kinetic by computer-assisted sperm analyzer, sperm penetration by cervical mucus penetration assay, and membrane integrity by hypo-osmotic swelling test. Sperm capacitation, apoptosis, and cell survival were analyzed using western immunoblotting.Statistical Analysis Used:One-way ANOVA on SPSS 21 combined with post hoc LSD test was used to analyze differences among the groups. A P < 0.05 was considered significant.Results:Sperm viability and kinetic were significantly reduced at H2O2 concentrations of 200 and 250 μM. H2O2 reduced sperm capability to penetrate cervical mucus and also damage cell membrane integrity at all concentrations used. H2O2 significantly inhibited sperm capacitation, indicated by reduced total tyrosine phosphorylation. H2O2 exposure stimulated activation of caspase 3 and significantly reduced phosphorylated AKT at all concentrations used.Conclusions:H2O2 comprehensively inhibits sperm qualities related to the capacity to fertilize oocyte, stimulates caspase activity, and inhibits cell survival.