The septin cytoskeleton is required for plasma membrane repair.

Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca2+-dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains.

Single Stage Reconstruction of Full Thickness Defects of Cheek with Pectoralis Major Myocutaneous Flap Using an Innovative Flap Technique.

The purpose of this article is to present an innovative technique in harvesting the pectoralis major myocutaneous flap (PMMC) which is very useful for reconstruction of post-ablative full thickness defects of cheek in elderly female patients. This is a prospective study of patients who underwent reconstruction with PMMC flap for post-ablative full thickness defects of the cheek and mandible in a tertiary cancer center over a period of 36 months. Sixty patients were included in the study. Mean duration of the hospital stay was 12 days. Eight patients (13.33%) had minor wound dehiscence, 6 (10%) had marginal skin necrosis of the flap, 2 (3.33%) patients had venous congestion, and 4 (6.66%) had wound dehiscence at the palate. Four (6.66%) patients had a gradual necrosis of the outer paddle. Our modification of elevation of the PMMC flap prevents its shearing which is especially useful in raising this flap in elderly female patients affected with advanced oral cancer.

Correction to: Single Stage Reconstruction of Full Thickness Defects of Cheek with Pectoralis Major Myocutaneous Flap Using an Innovative Flap Technique.

[This corrects the article DOI: 10.1007/s13193-021-01482-7.].

Virtual Laryngectomy Club: A Reality.

Total laryngectomy remains an essential treatment for locally advanced laryngeal carcinoma. However, it involves lifestyle changes for the patient, such as the inability to communicate verbally, breathing or aesthetic changes, which affect their quality of life and require comprehensive rehabilitation. Speech rehabilitation is of utmost importance for these patients. Hence to provide comprehensive rehabilitation after total laryngectomy we have been running a successful laryngectomee club at our institute for the benefit of our patients. But when the World Health Organization on March 11, 2020, declared the novel coronavirus (COVID-19) outbreak a global pandemic, many of our patients couldn't make their regular followup visits. We have successfully conducted the first ever online virtual meeting for our laryngectomee club members via video conferencing service.

Dermal metastases in oral cancer after curative treatment: a single institution cohort study.

Dermal metastasis (DM) is, by definition, the involvement of the skin by cancer cells that originate from cancer elsewhere in the body. The skin is considered a rare site of distant failure in head and neck cancer and DM is the bearer of a poor outcome. Literature about it is limited so this study was undertaken to analyse the factors associated with its incidence and outcomes. A prospectively maintained database on operated cases of oral cancer at a tertiary cancer centre was analysed, and patients who developed dermal metastases during follow up were evaluated. Factors that contributed to early DM and predicted survival after its development were studied. A total of 68 patients (2.8%) had DM as the first presentation of recurrence after a median disease-free period of five months. Early DM was significantly associated with skin involvement by the primary tumour at the time of presentation (p=0.06), extracapsular extension of nodes (p=0.004), and with those who required adjuvant chemotherapy in view of aggressive histology (p=0.021). Median (range) survival after the detection of DM was 97 (5-328) days (3.25 months). Surgical excision of isolated cases was associated with significantly increased survival after detection (p=0.05). Whenever it is feasible without too much morbidity, solitary DM should be excised.

Extraskeletal Ewing's Sarcoma Masquerading as Infantile Benign Neck Mass.

Despite being the second most common malignant bone tumor, Ewing's sarcoma remains uncommon in younger children and seldom seen in neonates and infants. Extraskeletal locations are even rarer, hardly ever suspected, and often misdiagnosed, causing delays in management. The histologic similarities of Ewing's sarcoma to more common pediatric small-blue-round-cell tumors such as lymphoma and neuroblastoma necessitate immunohistochemistry and molecular genetics for clinching the diagnosis. We report a soft-tissue Ewing's sarcoma in a 4-month-old female infant masquerading as a benign neck mass clinically, radiologically, cytologically, and intraoperatively. We also reviewed literature for any existing guidelines on when to biopsy neck masses in the pediatric population.

Primary Malignant Peripheral Nerve Sheath Tumor of the Trachea: a Case Report with Brief Review of Literature.

Malignant schwannoma, also called malignant peripheral nerve sheath tumor (MPNST), is a rare and aggressive tumor arising from the nerve sheath. We describe a rare case of endotracheal malignant peripheral nerve sheath tumor occurring in a middle-aged male who presented with asthma-like symptoms for 6 months with progressively increasing dyspnea. A computed tomogram (CT) scan of the thorax revealed near complete luminal obstruction of the trachea by a mass lesion at the level of the second and third tracheal rings. Microlaryngotracheoscopy revealed a fleshy pedunculated growth arising from the left side of the second and third tracheal rings and obliterating almost the entire tracheal lumen. Intraluminal complete excision of the mass was done. Later, he underwent excision of the 2nd and 3rd rings after the histopathology revealed MPNST. Patient after 28 months of follow-up is free of disease.

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Host cell perforation by listeriolysin O (LLO) activates a Ca2+-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing.

Host cell invasion is an indispensable step for a successful infection by intracellular pathogens. Recent studies identified pathogen-induced host cell plasma membrane perforation as a novel mechanism used by diverse pathogens (Trypanosoma cruzi, Listeria monocytogenes, and adenovirus) to promote their internalization into target cells. It was concluded that T. cruzi and adenovirus damage the host cell plasma membrane to hijack the endocytic-dependent membrane resealing machinery, thereby invading the host cell. We studied L. monocytogenes and its secreted pore-forming toxin listeriolysin O (LLO) to identify key signaling events activated upon plasma membrane perforation that lead to bacterial internalization. Using various approaches, including fluorescence resonance energy transfer imaging, we found that the influx of extracellular Ca2+ subsequent to LLO-mediated plasma membrane perforation is required for the activation of a conventional protein kinase C (cPKC). cPKC is positioned upstream of Rac1 and the Arp2/3 complex, which activation leads to F-actin--dependent bacterial internalization. Inhibition of this pathway did not prevent membrane resealing, revealing that perforation-dependent L. monocytogenes endocytosis is distinct from the resealing machinery. These studies identified the LLO-dependent endocytic pathway of L. monocytogenes and support a novel model for pathogen uptake promoted by plasma membrane injury that is independent of membrane resealing.

High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair.

The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.