Basal Cells Research Articles

Pathological Mechanisms Involved in Epidermolysis Bullosa Simplex: Current Knowledge and Therapeutic Perspectives.

Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of mechanobullous diseases characterized by non-scarring blisters and erosions on the skin and mucous membranes upon mechanical trauma. The simplex form (EBS) is characterized by recurrent blister formation within the basal layer of the epidermis. It most often results from dominant mutations in the genes coding for keratin (K) 5 or 14 proteins (KRT5 and KRT14). A disruptive mutation in KRT5 or KRT14 will not only structurally impair the cytoskeleton, but it will also activate a cascade of biochemical mechanisms contributing to EBS. Skin lesions are painful and disfiguring and have a significant impact on life quality. Several gene expression studies were accomplished on mouse model and human keratinocytes to define the gene expression signature of EBS. Several key genes associated with EBS were identified as specific immunological mediators, keratins, and cell junction components. These data deepened the understanding of the EBS pathophysiology and revealed important functional biological processes, particularly inflammation. This review emphasizes the three EBS subtypes caused by dominant mutations on either KRT5 or KRT14 (localized, intermediate, and severe). It aims to summarize current knowledge about the EBS expression profiling pattern and predicted molecular mechanisms involved and to outline progress in therapy.

Human efferent ductules and epididymis display unique cell lineages with motile and primary cilia.

Previous research has illustrated the role of cilia as mechanical and sensory antennae in various organs within the mammalian male reproductive system across different developmental stages. Despite their significance in both organ development and homeostasis, primary cilia in the human male reproductive excurrent duct have been overlooked due to limited access to human specimens. This study aimed to characterize the unique cellular composition of human efferent and epididymal ducts, with a focus on their association with primary cilia. Human efferent ductules/epididymides from five donors aged 32-47 years, were obtained through our local organ transplant program. Cell lineage specificity and primary cilia features were examined by immunofluorescent staining and confocal microscopy in the efferent ductules and the distinct segments of the epididymis. The epithelium of the human efferent duct exhibited estrogen receptor-positive cells with primary cilia, FoxJ1-positive multiciliated cells with numerous motile cilia, and non-ciliated intraepithelial immune cells. Notably, intraluminal macrophages, identified by CD163/CD68 positivity, were observed to engage in sperm phagocytosis. In all three segments of the human epididymis, primary cilia were found on the surface of principal and basal cells. Our research indicates that the human efferent ductules create a distinct environment, characterized by the presence of two types of ciliated cells that are in contact with immune cells. The discovery of sensory primary cilia exposed on the surface of reabsorptive cells in the efferent ductules, as well as on basal and principal cells in the epididymis, lays the foundation for complementary functional studies. This research uncovers novel characteristics exclusive to human efferent ductules and epididymides, providing a basis for exploring innovative approaches to male contraception and infertility treatment.

SP-101, A Novel Adeno-Associated Virus Gene Therapy for the Treatment of Cystic Fibrosis, Mediates Functional Correction of Primary Human Airway Epithelia From Donors with Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Although CF affects multiple organs, lung disease is the main cause of morbidity and mortality, and gene therapy is expected to provide a mutation-agnostic option for treatment. SP-101 is a recombinant adeno-associated virus (AAV) gene therapy vector carrying a human CFTR minigene, hCFTRΔR, and is being investigated as an inhalation treatment for people with CF. To further understand SP-101 activity, in vitro studies were performed in human airway epithelia (HAE) derived from multiple CF and non-CF donors. SP-101 restored CFTR-mediated chloride conductance, measured via Ussing chamber assay, at a multiplicity of infection (MOI) as low as 5E2 in the presence of doxorubicin, a small molecule known to augment AAV transduction. Functional correction of CF HAE increased with increasing MOI and doxorubicin concentration and correlated with increasing cell-associated vector genomes and hCFTRΔR mRNA expression. Tropism studies using a fluorescent reporter vector and single-cell mRNA sequencing of SP-101-mediated hCFTRΔR mRNA demonstrated broad expression in all cell types after apical transduction, including secretory, ciliated, and basal cells. In summary, SP-101, particularly in combination with doxorubicin, shows promise for a novel CF treatment strategy and strongly supports continued development.

Eco-friendly synthesis of Co3O4 nanoparticles using Millettia pinnata towards increased anti-oxidant, anti-biofilm and cytocompatibility properties against biofilm producing bacteria and human pulmonary alveolar basal cells

Biofilm architecture is an emerging threat worldwide due to enhanced antibiotic resistance against existing antibiotics, thus heightening the need to discover the alternative therapies that can eradicate the biofilm colonization. Current study highlights the eco-friendly, non-toxic nature of Co3O4 nanoparticles (NPs) using Millettia pinnata (M. pinnata) leaves extract and their application in anti-oxidants, anti-bacterial, anti-biofilm and cytocompatibilities. The GC-MS and FT-IR spectral data suggested that phytochemical derivatives of M. pinnata leaf extract simultaneously contributed in bio-reduction process and capping agent of Co3O4 NPs, which might be reason for anti-oxidant activities. Further, XRD analysis confirms the polycrystalline behaviour of Co3O4 NPs having hexagonal crystal system. FESEM, HRTEM, EDAX and elemental mapping revealed the formation of agglomerated granular shape along with nanorods on the surface of Co3O4 NPs, which confirms the presence of carbon and oxygen. Additionally, the characteristic band and thermal decomposition of Raman and TGA spectral data confirmed the molecular structure of Co3O4 NPs. Subsequently, both the M. pinnata leaves extract and Co3O4 NPs were found to be excellent source of antioxidants and produced significant DPPH and hydroxyl scavenging activities. Furthermore, the sub-biofilm inhibitory concentration of biosynthesized Co3O4 NPs reduced the biofilm growth in S. aureus and K. pneumoniae by crystal violet assay at increasing concentration. In addition, the membrane integrity of biofilm damages and architecture destruction was evidently proved due to the biosynthesized Co3O4 NPs by confocal laser scanning electron microscope and scanning electron microscope observation. Finally, the cytotoxicity experiment results indicated, biosynthesized Co3O4 NPs has no toxicity against human pulmonary alveolar epithelial cells at maximum concentration. Accordingly, findings of this study supported the utility of safety and efficiency of biosynthesized Co3O4 NPs against biofilm producing bacteria, and deserve as promising future drug discovery target for various bacterial infections.

DERMOSCOPIC FEATURES OF BASAL CELL CARCINOMA: SYSTEMATIC REVIEW

Abstract Background: Basal cell carcinoma is one of the most common cancers worldwide, it is a locally destructive and slowly spreading tumor arising from the epidermis or hair follicles, in which the peripheral cells are similar to the cells of the basal layer of the epidermis and which rarely metastasize. Despite its slow growth and low potential for metastasis, basal cell carcinoma (BCC) is one of the most pressing problems in dermato-oncology due to its high incidence rates, variety of clinical forms and frequent relapses after any treatment methods. Currently, the most accessible non-invasive method for examining skin tumors is dermatoscopy. Aim: The purpose of this review is to highlight the dermoscopic features encountered in basal cell carcinoma and to outline the role of dermoscopy for diagnosis this cancer. Methodology: The systematic review included articles from Google Scholar, Medline, Scopus, Web Of Sciences, PubMed was conducted. For the search, the following keywords were included: dermoscopy of pigmented form of basal cell carcinoma; dermoscopy of nonpigmented form of basal cell carcinoma. Results: Conducted a preliminary search and reviewed 62 titles and abstracts in this review and 27 full-text articles were selected of high methodological quality. Dermoscopy is a non-invasive technique, which allows early diagnosis based on the presence of typical vascular structures, pigmented structures, and ulceration and the absence of specific melanocytic structures. However, the use of dermoscopic technique is limited by the lack of prognostically significant dermatoscopic criteria confirming the different morphological nature of tumor growth. Conclusion: The current systematic review demonstrated, based on the observed features, this method allows for differential diagnosis of malignant and benign, as well as melanocytic and non-melanocytic skin neoplasms, as well as diagnosis of tumors at the earliest stage of their development.

Establishing a Mouse Model of Surgical Vocal Fold Injury.

Animal models of vocal fold (VF) surgical injury and scar formation provide insight into the wound healing process. The purpose of this study was to establish an alternative model of surgical injury to the mouse VF using materials commonly available in most research laboratories or for purchase and to investigate wound healing of the epithelium (EP) and lamina propria (LP). Mice were anesthetized by isoflurane gas delivery and positioned on a platform so that the larynx could be observed using a laryngoscope and dissection microscope. Unilateral VF injury was created using a wire brush. Mice were euthanized and the larynx evaluated 1-, 3-, 5-, 7-, 14-, and 28-days following injury. Histological and immunofluorescent analysis was used to evaluate thickness of the EP, LP area, proliferative (Ki67+) and basal cells (p63+) in the EP, and collagen III content in the LP. The depth of injury reached the superficial thyroarytenoid muscle on Day 1. The thickness of the EP of the injured VF was increased on Days 3 and 5, and the LP area was increased on Days 3, 5, and 7 as compared with the uninjured VF. Ki67+ and p63+ cells were increased on Day 3 and collagen III content was increased on Days 5 and 28 as compared with the uninjured VF. We successfully established an alternative method of creating unilateral VF injury in the mouse. This method will be useful for future research regarding VF surgical injury and wound healing. N/A Laryngoscope, 2024.

First Report of Leaf Blight of Ginkgo biloba L. Caused by Neopestalotiopsis clavispora G.F. Atk. in Sichuan, China.

Ginkgo (Ginkgo biloba L.), the oldest existing tree species in the world, is an important ornamental and medicinal plant, widely planted in China. In October 2022, a new leaf blight disease was observed in Chengdu city (30°05'to 31°26'N, 102°54'to 104°53'E). Disease incidence averaged 82.5% across five foci. The typical symptomatology begins when leaf margins turn yellow and small black spots appear at the edge of the leaf, chlorotic areas turn brown, dry and deformed. Gradually, the necrotic lesions spreads to the middle of the leaf and eventually the whole leaf falls off. Infected tissues from ten leaves were cut into small pieces (5 × 5 mm); surface sterilized for 30 s in 3% sodium hypochlorite; 60 s in 75% ethanol; rinsed three times in sterile water; placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL); and incubated at 25°C for 3 to 8 days. A hyphae was removed from the edge of the fungal colony and placed onto potato dextrose agar (PDA) plates. After incubation at 25℃ with a 12-hour light/dark cycle for 8 days, the colony diameter reached 77.5 to 81.5 mm. Colonies grown on PDA were white, cotton, flocculent, undulating on the surface, dense in aerial hyphae and light yellow on the back. Black pycnidia formed superficially, scattered over the PDA, following two weeks of incubation. Pycnidia contained sticky black conidia. The spores were were spindle shaped, with five cells, and four septations measuring 20.9 to 34.8 µm × 6.8 to 8.8 µm (avg. 28.4 × 7.6 µm; n=40). The three median cells were versicolored, typically two dark brown cells and one light brown cell, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (2.87 to 4.1 µm long; n = 40) and two to three apical appendages (18.3 to 29.1 µm long; n = 40). Based on colony and conidial morphology, the isolate was identified as N. clavispora (Maharachchikumbura et al. 2014). The partial sequence of the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using the universal primer pairs ITS1/ITS4(Zhang et al. 2022), BT2A/BT2B (Li Yuan et al. 2022), and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. Sequences of representative isolate LQYX were deposited in GenBank (ITS: OQ152504, TUB: OQ168328, and TEF1: OQ168329). BLAST results indicated that the ITS, TUB, and TEF1-α sequences showed 99 to 100% identity with N. clavispora sequences at NCBI (GenBank MG729689, MG740735, and MG740758). Identification was confirmed by Bayesian inference using Mr. Bayes. Next, inoculations were conducted on leaves of ten G. biloba in the field to verify the pathogenicity of LQYX. Ten healthy leaves of each plant were surface sterilized with 75% ethanol, and the wound was rubbed out on the leaf edge on the sterilized sanding paper. A conidia suspension (1 × 107 ml-1) was sprayed on the leaves, aseptic water was used as the control, and the transparent plastic bag was used to maintain relative humidity. After 14 days (26 ℃, 14 hours light / 10 hours dark), the inoculated leaves had similar symptoms as the original diseased plants, whereas controls were asymptomatic. The N. clavispora was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf blight of G. biloba caused by N. clavispora in China, which has greatly affected the appearance of the city and should be further studied. This report can help identify this disease and further develop effective control measures.

Skin Absorbed Dose Coefficients for Human Legs from Beta Radiation as a Function of Height

External exposure to skin from beta-emitter radionuclides following severe reactor accidents or nuclear testing can result in beta burning and other health complications. The skin absorbed dose coefficient (SADC) measures the energy deposition into the skin during such accidents. The U.S. Environmental Protection Agency has published several reports to measure the possible energy deposition into the skin in such accidents. However, the most recent SADC published by Federal Guidance Report (FGR) 12 was computed only at one meter above the contaminated surface. Therefore, it was necessary to develop a model to estimate the absorbed dose coefficients for skin at different heights. In this manuscript, Geant4, a Monte Carlo simulator toolkit, was used to estimate the absorbed dose coefficients from electron sources located on the soil surface with energies ranging from 0.1 to 4 MeV. The energy deposited from primary electrons, secondary electrons, and photons in a 50 µm thick layer of epidermis tissue (Basal Cells Layer) located at a depth of 50 µm from the skin surface was estimated at several discrete heights of human leg phantom. More than 40% of the total energy deposited comes from secondary electrons and photons in energy sources of 0.1 and 0.2 MeV on average, but for higher energies, this percentage is less than 1%, which indicates primary electrons are the main source of the deposited energy in the skin. Furthermore, the results showed the energy deposited into skin closer to the ground was 50–100% higher than the previously estimated doses for 1 m above the ground. The results from Geant4 showed a great correlation (R2 = 0.972) with the FGR 12 data at one meter height, and they were aligned with the published values from FGR 12, which validated the simulation results. Therefore, the calculated dose coefficients for different energy sources and different heights could be used in radiation protection measurements.

Cowper Glands Identified in Prostate and Urethral Specimens: A Comprehensive Immunohistochemical Characterization and Potential Diagnostic Pitfall.

Cowper glands recognition remains one of the key histoanatomic benign mimics of prostatic adenocarcinoma. In most instances, these can be identified based on the dimorphic population of lobulated acini and duct(s). However, in the prostate biopsy setting with incomplete/distorted cores, this may not be immediately apparent and may warrant use of immunohistochemistry to argue against prostatic adenocarcinoma. Although immunohistochemical pitfalls in Cowper glands have been described, to our knowledge a comprehensive evaluation of both traditional and purportedly prostate-specific novel markers in Cowper glands has not been previously performed. Herein, we studied the clinicopathological and immunohistochemical features of 21 male patients (age range 39-81 years; mean = 63 years), including 15 prostate biopsies (7 of which also had prostate cancer in the same specimen set and 2 of which had both prostate cancer and Cowper glands in the same biopsy core). Immunohistochemistry showed the following results in Cowper glands: 100% positive for NKX3.1, 100% positive (basal cells) for both high molecular weight keratin and p63, 57% positive for PSAP, 25% positive for PSMA, 5% positive for AMACR, and 0% positive for PSA. In conclusion, for specimens lacking appreciable dimorphic morphology, caution should be rendered when using prostate-specific markers (PSA, PSAP, PSMA, and NKX3.1) as these can show considerable staining in Cowper glands and be a pitfall. Instead, findings from this cohort indicate relying on basal markers (high molecular weight keratin/p63; either individually or in a "cocktail" approach) and PSA are most useful in distinguishing Cowper glands (retained basal cell markers staining) from prostatic adenocarcinoma.

Changes of epidermal proteins immunolocalization in the corneous layer from embryonic to definitive avian beak

The process of keratinization and cornification in the developing beak has been studied through immunofluorescence and immunogold electron microscopy in chick and zebrafinch embryos. After the curved beak anlagen appears at the tip of the maxillar bone, 5–8 layers of embryonic epidermis are generated from the basal layer of the epidermis. These cells are weakly immunoabeled for IFKs (Intermediate Filament Keratins) and more intensely for scaffoldin, a protein of the EDC (Epidermal Differentiation Complex) involved in the soft keratinization of the embryonic epidermis. Immunolabeling for CBPs (Corneous Beta Proteins) is visible in the transitional embryonic layers that are temporarily generated between the embryonic and definitive beak epidermis. The electron microscope reveals that intermediate layers contain immunolabeled periderm granules for scaffoldin mixed with bundles of corneous material immunolabeled for CBPs. Intense CBPs labeling occurs in the compacting corneous bundles of beta-keratinocytes in the definitive beak while scaffolding labeling disappears. The embryonic epidermis is sloughed before hatching. Sox (Sulfhydryl Oxidase) immunolabeling reveals that the enzyme is almost absent in embryonic layers but is present in transitional and definitive beta-keratinocytes. This indicates the formation of cross-linked disulfide bonds in the definitive corneous layer of the beak. Some calcium precipitation, suggested from von Kossa staining, occurs in the corneous layers only on the 18th day of development in the chick, in preparation for hatching.

Dynamic regulation of tissue fluidity controls skin repair during wound healing

During wound healing, different pools of stem cells (SCs) contribute to skin repair. However, how SCs become activated and drive the tissue remodeling essential for skin repair is still poorly understood. Here, by developing a mouse model allowing lineage tracing and basal cell lineage ablation, we monitor SC fate and tissue dynamics during regeneration using confocal and intravital imaging. Analysis of basal cell rearrangements shows dynamic transitions from a solid-like homeostatic state to a fluid-like state allowing tissue remodeling during repair, as predicted by a minimal mathematical modeling of the spatiotemporal dynamics and fate behavior of basal cells. The basal cell layer progressively returns to a solid-like state with re-epithelialization. Bulk, single-cell RNA, and epigenetic profiling of SCs, together with functional experiments, uncover a common regenerative state regulated by the EGFR/AP1 axis activated during tissue fluidization that is essential for skin SC activation and tissue repair.

Protective Effects of Chitosan-Loaded Pomegranate Peel Extract Nanoparticles on Infertility in Diabetic Male Rats.

Diabetes Mellitus (DM) is known to have an impact on the health of the male reproductive system. It is linked to low sperm quality, increased oxidative stress, and an increased generation of reactive oxygen species in the seminal fluid. Pomegranate extract has phenolic compounds and significant protective properties against oxidative stress, male sex hormone disruptions, and sperm abnormalities. The current study aimed to evaluate the effectiveness of Pomegranate Peel Extract Nanoparticles (PPENPs) on male fertility in diabetic rats. DM was induced in rats by intraperitoneal injection of streptozotocin (60 mg/kg). Twenty-four rats were divided into four groups, 6 rats in each group: control, DM, DM+empty NPs (60 mg/kg, orally), and DM+PPENPs (60 mg/kg, orally). Administration of PPENPs increased the levels of insulin, FSH, LH, testosterone, catalase, glutathione reduced, and semen fructose. PPENPs also improved sperm quality, as seen by improvements in sperm morphology, motility, count, and the ability of metabolically active spermatozoa to convert blue resazurin dye to pink resorufin. However, PPENPs decreased levels of glucose, malonaldehyde, nitric oxide, and sperm abnormalities. Also, histological investigation of the PPENPs showed improvement in testis tissue architecture and increased the diameter size of seminiferous tubules and germinative layer thickness. Our investigation proved that the treatment of PPENPs has a protective effect on the reproductive system of male diabetic rats, improving fertility parameters, healthy sperm profiles, and the antioxidant system.

Pre-Descemet Corneal Dystrophy – Changes in Corneal Morphology in Confocal Microscopy

PurposeTo identify and describe the in-vivo microstructural changes in the cornea of patients with pre-Descemet’s membrane corneal dystrophy.MethodsTen patients (20 eyes) were examined in the Laser Microsurgery Centre in Warsaw. The ophthalmic examination, including visual acuity and slit lamp biomicroscopy, was carried out. Because of suspicion of corneal dystrophy, in vivo confocal microscopy of the cornea was also performed.ResultsBiomicroscopy revealed bilateral, dust-like fine opacities in the deep parts of the corneal stroma. Confocal microscopy showed pleomorphic structures (enlarged keratocytes) containing dense, hyperreflective, granular inclusions in the posterior stroma, next to the Descemet’s membrane. In 3 patients (6 eyes) punctate particles were seen extracellularly also in the mid stroma. Superficial and basal epithelial layers, anterior stroma and endothelium cells appeared normal.Conclusions1. In vivo confocal microscopy findings of cornea with pre-Descemet’s membrane dystrophy are characteristic. 2. In vivo confocal microscopy is a useful tool in diagnosing rare corneal dystrophies in cases where classical examination methods give inconclusive results.

Burn Wound Healing Activity of Hydroxyethylcellulose Gels with Different Water Extracts Obtained from Various Medicinal Plants in Pseudomonas aeruginosa-Infected Rabbits.

Burn injuries represent a significant problem in clinical practice due to the high risk of infection and the prolonged healing process. Recently, more attention has been given to natural remedies such as water extracts of various medicinal plants, which possess anti-inflammatory and wound healing properties. The aim of this study is to evaluate the efficacy and safety of Satureja montana L. and other water extracts in a burn wound model. The study involved male Californian rabbits (n = 52) divided into eight groups. Burn wounds were modeled on the animals and subsequently treated with gels based on Satureja montana L. and other water extracts. The reparative potential of the epidermis (assessed by Ki-67 expression), the state of local immunity (measured by the number of CD-45 cells), and the anti-inflammatory role of mast cells (measured by tryptase levels) were evaluated. Bacteriological and morphological studies were conducted. The most pronounced bactericidal, reparative, and immunostimulatory effects were observed after the treatment using a gel mixture of water extracts from Satureja montana L., Salvia sclarea, Coriandrum sativum L., and Lavandula angustifolia in equal proportions (1:1:1:1). The other gels also demonstrated high efficacy in treating burn wounds, especially when using a strain of Pseudomonas aeruginosa resistant to several antibiotics. Immunohistochemical studies showed a significant increase in the number of Ki-67-positive cells in the basal layer of the epidermis and a decrease in the number of CD-45-positive cells, indicating improved proliferative activity and reduced inflammation. This study confirms the hypothesis that the use of water extract mixtures significantly enhances the reparative potential, improves the immune response in the treatment of burns, and promotes wound healing. These findings pave the way for further research and the application of complex phytotherapeutic agents, specifically water extracts of medicinal plants containing phenols and antioxidants in burn wound therapy.

A nasal airway-on-chip model to evaluate airflow pre-conditioning during epithelial cell maturation at the air-liquid interface

The function of a well-differentiated nasal epithelium is largely affected by airflow-induced wall shear stress, yet fewin vitromodels recapitulate this dynamic condition. Models which do expose cells to airflow exclusively initiate flow after the differentiation process has occurred.In vivo, basal cells are constantly replenishing the epithelium under airflow conditions, indicating that airflow may affect the development and function of the differentiated epithelium. To address this gap in the field, we developed a physiologically relevant microphysiological model of the human nasal epithelium and investigated the effects of exposing cells to airflow during epithelial maturation at the air-liquid interface. The nasal airway-on-chip platform was engineered to mimic bi-directional physiological airflow during normal breathing. Primary human nasal epithelial cells were seeded on chips and subjected to either: (1) no flow, (2) single flow (0.5 dyne cm-2flow on Day 21 of ALI only), or (3) pre-conditioning flow (0.05 dyne cm-2on Days 14-20 and 0.5 dyne cm-2flow on Day 21) treatments. Cells exposed to pre-conditioning showed decreased morphological changes and mucus secretions, as well as decreased inflammation, compared to unconditioned cells. Our results indicate that flow exposure only post-differentiation may impose acute stress on cells, while pre-conditioning may potentiate a properly functioning epitheliumin vitro.

Neopestalotiopsis rosae, a novel pathogen causing leaf blight and crown rot of strawberries in India

Neopestalotiopsis have been reported to affect sustainable strawberry production across the world. Strawberry leaves with leaf blight symptoms were collected in January 2023 from the nurseries and fields of Department of Horticulture, Gandhi Krishi Vigyan Kendra, GKVK, Bangalore, India. Black conidiomata with slimy black conidial mass was produced upon incubating at 28 ± 2 °C. Conidia were fusoid to ellipsoidal, and comprised five cells. The middle three cells were versicolored. Whereas, the apical and basal cells harboured three to four and one appendages, respectively. Neopestalotiopsis rosae was confirmed based on sequencing of internal transcribed spacer (ITS) region of rDNA, β-tubulin (TUB2) and translation elongation factor 1-alpha (EF1-α) gene regions and phylogenetic analysis. Pathogenicity tests confirmed that Neopestalotiopsis rosae as the inciting agent of leaf blight and crown rot disease in strawberry posing a threat to strawberry production in India.

Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-Length CFTR cDNA in Airway Stem Cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

A novel immunofluorescence study of Lingual Salivary Glands in the Egyptian Tortoise (Testudo kleinmanni) and its ecological significance

The Egyptian tortoise (Testudo kleinmanni) is remarkably adapted to its harsh desert environment, a characteristic that is crucial for its survival under extreme conditions. This study was aimed at providing a deeper understanding of the lingual salivary gland structures in the Egyptian tortoise and examining how these structures help the tortoise manage hydration and nutrition in arid conditions. Utilizing a combination of light microscopy and immunofluorescence, this research introduced pioneering methods involving seven different antibodies, marking a first in the study of reptilian salivary glands. Our investigations categorized the tortoise’s salivary glands into papillary and non-papillary types. The papillary glands were further classified into superficial, deep, interpapillary, and intraepithelial salivary glands, while non-papillary glands included superficial and deep lingual types. Structurally, these glands are organized into lobules, delineated by interlobular septa, and are equipped with a duct system comprising interlobular, intercalated, and main excretory ducts with gland openings on the tongue's surface and the papillae surfaces. Notably, the superficial glands displayed both tubuloalveolar and acinar configurations, whereas the deep lingual glands were exclusively acinar. Immunofluorescence results indicated that α-smooth muscle actin (α-SMA) was prevalent in myoepithelial cells, myofibroblasts, and blood vessels, suggesting their integral role in glandular function and support. E-cadherin was predominantly found in epithelial cells, enhancing cell adhesion and integrity, which are critical for efficient saliva secretion. Importantly, Mucin 1 (MUC1) and Mucin 5B (MUC5B) staining revealed that most glands were mucous in nature, with MUC5B specifically marking mucin within secretory cells, confirming their primary function in mucous secretion. PDGFRα and CD34 highlighted the presence of telocytes and stromal cells within the glandular and interlobular septa, indicating a role in structural organization and possibly in regenerative processes. Cytokeratin 14 expression was noted in the basal cells of the glands, underscoring its role in upholding the structural foundation of the epithelial barrier. In conclusion, this detailed morphological and immunological characterization of the Egyptian tortoise’s salivary glands provides new insights into their complex structure and essential functions. These findings not only enhance our understanding of reptilian physiology but also underline the critical nature of salivary glands in supporting life in arid environments. This study's innovative use of a broad range of immunofluorescence markers opens new avenues for further research into the adaptive mechanisms of reptiles.

Effect of short-term fasting on the cisplatin activity in human oral squamous cell carcinoma cell line HN5 and chemotherapy side effects

BackgroundKetogenic interventions like short-term fasting show potential as complementary therapies to enhance the effectiveness of chemotherapy for cancer. However, the specific effects of fasting on head and neck squamous cell carcinoma (HNSCC) cells and healthy oral mucosa cells during these treatments are not well understood. This study investigates whether short-term fasting can differentially impact HNSCC cell survival and viability compared to healthy keratinocytes while undergoing standard chemotherapy regimens.MethodsThis study investigated the effects of fasting on cell viability in HN5 cell line and healthy oral keratinocyte cells. The HN5 cell line, derived from human tongue squamous cell carcinoma, and primary human keratinocytes isolated from the basal layer of gingival epithelium were divided into three groups: (1) control, (2) treated with the standard chemotherapeutic agent cisplatin, and (3) treated with cisplatin under fasting conditions achieved through 48-hour glucose restriction mimicking the blood glucose levels of fasted individuals. Cell proliferation was assessed at 48 and 72 h using the MTT assay, a colorimetric method based on mitochondrial dehydrogenase activity. Flow cytometry analysis with specific apoptosis and necrosis markers distinguished between early and late apoptotic, necrotic, and viable cells.ResultsCell viability in HN5 and healthy keratinocyte cells decreased in cisplatin with low glucose groups compared to cisplatin and control groups. The same results were observed for healthy keratinocyte cells; only a decrease in cell viability in cisplatin groups compared to control groups was observed, which was not statistically significant. Cell apoptosis in HN5 and healthy keratinocyte cells increased in cisplatin with low glucose groups compared to cisplatin and control groups. In healthy keratinocyte cells, the cisplatin with low glucose group showed an impressive increase in necrosis, late apoptosis, and early apoptosis and a significant decrease in live cells compared with other groups.ConclusionThis study revealed that short-term fasting chemotherapy significantly improved HNSCC cell line apoptosis and necrosis.