Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Genome sequence of PSonyx, a singleton bacteriophage infecting Corynebacterium glutamicum.

PSonyx is a newly isolated phage that infects Corynebacterium glutamicum. This siphovirus was isolated from a French pond in the south of Paris by students from Paris-Saclay University. Its 80,277-bp singleton genome carries 136 protein-coding genes and 5 tRNAs.

Removal of Unwanted Hair: Efficacy and Safety of 755-nm Alexandrite Laser Equipped with a 30 mm Spot Handpiece.

Background: Alexandrite 755 nm laser has been currently recognized one of the gold standards for the permanent hair removal business because of its sufficient tissue penetration and higher affinity for melanin compared with Nd:YAG, making the treatment more incisive and effective. Objective: In this study, we evaluated an Alexandrite 755 nm laser with a 30-mm spot for a new and fast hair removal treatment. Methods: Patients of both genders, with skin Fitzpatrick's types, ranging from II to III, were asked to avoid any epilation techniques 4 weeks before the laser hair removal session. Participants underwent a minimum of three to a maximum of eight sessions of treatment, every 6 weeks. Six months after the last treatment, the final evaluation was completed. The treated body areas included arms, legs, axillae, and trunk. The subjects used a numeric scale with a range of 0 to 10 (0: no pain, 10: unbearable), to indicate the associated pain level experienced. By comparing the percentage of terminal hairs before and after laser treatment, hair reduction was determined. Results: The patients reported only slight post-treatment erythema that disappeared after a few hours and only two patients showed persistent erythema lasting a few weeks. The pain was found to be 4.9 ± 0.9 after single pass emission. Conclusions: The 755 nm laser with the new handpiece has proven to be an ideal wavelength for hair removal, allowing efficient, safe, and faster treatments to be performed for the patient.

Novel Management of Granuloma Formation Secondary to Dermal Filler with Intralesional 1444 nm Nd:YAG Laser Technique.

Background: Dermal fillers for soft tissue for the treatment of face sagging, volume loss, and wrinkles have become popular among patients of all ages and ethnicities, and their use is becoming increasingly widespread. Aim: the goal of this study was to evaluate the effectiveness and safety of a micro-pulsed, 1444 nm Nd:YAG laser on dermal filler complications, in particular on granuloma management. Methods: A subcutaneous, 1444 nm Nd:YAG laser was used on five female patients (range age 52-68 years) with hyaluronic filler granulomas located on the face (two on the cheek area and three on the lips); three patients had self-injected the filler, buying it online. Before and after the therapy, the patients received a skin ultrasound to determine the form and location of the granulomas and to determine if there had been a full or partial resolution. During this study, all possible adverse effects at the treatment site were monitored. The 5-point Global Aesthetic Improvement Scale (GAIS) (0 point-no change; 1 point-25%, mild improvement; 2 points-50%, moderate improvement; 3 points-75%, good improvement; 4 points-100%, excellent improvement) was recorded at a 3-month follow-up. Results: good results were obtained in the treatment of filler granulomas with the intralesional 1444 nm laser, even if just a single treatment was performed (one intervention was effective for curing granulomas up to 5 mm in diameter). Three patients were satisfied with excellent improvement, and two patients experienced good improvement. The results are functional and aesthetically satisfying, as shown by photographic assessment. At the last follow-up, the granuloma had reduced or completely disappeared in all cases, and no infections, burns, scarring or fibrosis, episodes of severe bleeding, or other serious adverse effects had been reported. All subjects tolerated the post-treatment period well. Conclusions: Our findings showed that granuloma treatment with an intralesional 1444 nm Nd:YAG laser is a minimally invasive, easy, fast, efficient, and low-risk procedure.

Microwave Therapy for Frostbite Management: A Case Series.

Microwave body tightening and contouring is a common and efficient cosmetic medical procedure. The current study presents preliminary data about microwave treatment for body contouring where an innovative and unexpected benefit on frostbites was shown. This is a case series on two patients with frostbite treated with microwave therapy. The participants received the treatment for five sessions at 20-day intervals, including the beginning of the study. In addition to being satisfied with the treatment of their skin imperfections, the patients noticed a remarkable and progressive improvement in frostbite on their limbs. Both patients experienced a significant improvement in skin sensation and appearance and no side effects were observed. Our findings confirmed the safety and efficacy of microwave therapy in treating cellulite and skin laxity but rather a positively effect and a significant improvement in the treatment of frostbite as a secondary intention.

Ex Vivo Human Histology Fractional Treatment with a New CO2 Scanner: A Potential Application on Deep Scarring.

Background and Objectives: For many years, fully ablative laser treatments, particularly those performed with a carbon dioxide (CO2) laser, were regarded as the gold standard for resurfacing. This study's goal is to assess the depth that can be reached by a new CO2 scanner system, through a skin model with greater dermal thickness, to use in the treatment of deep scarring. Materials and Methods: Male human skin tissue was laser-treated using a CO2 fractional laser and a new scanning system, and all samples were fixed in 10% neutral buffered formalin, dehydrated using a series of crescent alcohol, embedded in paraffin, sectioned in series (4-5 µm thick), stained with haematoxylin and eosin (H&E), and then analysed under an optical microscope. Results: From the epidermis through the underlying papillary and reticular dermis to various depths of the dermis, microablation columns of damage and coagulated microcolumns of collagen were observed. The reticular dermis was fully penetrated up to 6 mm at higher energy levels (210 mJ/DOT), resulting in deeper tissue injury. Although the laser might penetrate further, the skin stops there, leaving just the fat and muscular tissue. Conclusions: The deep layers of the dermis can be penetrated by the CO2 laser system throughout the entire dermal thickness when using the new scanning system, suggesting that this laser's potential impact, at the selected settings, covers all skin targets required to perform superficial or deep treatments on any dermatological issue. Finally, patients who have problems, such as morbid scar-deep complications, which affect their quality of life, are more likely to profit from this innovative technique.

Laser emission at 675 nm: In vitro study evidence of a promising role in skin rejuvenation.

Light-based therapies have been proven to influence and perhaps reverse skin ageing at clinical, molecular and histologic levels. Laser technology decreases photodamage by promoting collagen type I and III synthesis and enhancing the expression of heat shock protein. Aims: This study aims to assess different doses of 675 nm irradiation on human dermal fibroblast cells to evaluate the potential therapeutic effects on the rejuvenation process. Methods: This study employed a laser system that emits 675 nm wavelength: 260, 390, 520 and 650 J/cm2 doses were tested on adult human dermal fibroblast cells. Cellular viability, proliferation, and synthesis of type I and III collagen were studied. Results: No dose tested showed effects on cell viability and proliferation at 24 and 48 h from the irradiation. Doses of 260 and 520 J/cm2 causes a significant decrease in type I collagen fluorescence intensity, while 390 J/cm2 elicits a significant increase in type III collagen expression. Conclusions: Our results showed that 675 nm laser irradiation does not affect cell viability while modulating cell proliferation and collagen synthesis in human adult cultured fibroblasts in vitro. These findings suggest that 675 nm laser irradiation potentially plays a role in skin rejuvenation.

Minimally invasive 1,444-nm Nd:YAG laser treatment for axillary bromhidrosis.

Background: Axillary bromhidrosis is an apocrine glands hyperactivity disease. Methods: A total of 24 patients (15 men and 9 women) with axillary bromhidrosis underwent a laser procedure with a 1,444-nm Nd:YAG laser. Parameters evaluated in this study were as follows: the degree of malodor (T0, baseline; T30, after 1 month; and T180, after 6 months), postoperative pain, short-term decreased mobility (T1, after 1 day; T7, after 7 days; and T30, after 1 month), and overall satisfaction (T30, after 1 month and T180, after 6 months). A visual analog scale (VAS), from 0 to 10, was used to assess pain and decreased mobility, with lower values denoting less severity. Results: A total of 24 patients were followed up for 6 months after laser treatment. At baseline, all patients (100%) complained of a strong axillary malodor (mean degree of malodor at T0 = 2.0 ± 0.00). It decreased to 0.50 ± 0.64 at T30. At T180, the degree of malodor was 0.54 ± 0.57. Both T30 and T180 degrees of malodor significantly decreased from the baseline value (p < 0.01). The mean degree of patient satisfaction at T30 was 1.75 ± 0.52, and at T180, it was 1.67 ± 0.21. Among the 24 patients, eight complained of moderated pain 1 day after treatment. The pain subsided on day 7, except for two patients, with VAS = 1. Pain and mobility restrictions were in any case resolved within T30. Conclusion: Treatment with a 1,444-nm Nd:YAG laser for subdermal interstitial coagulation could be a less invasive and more effective option treatment for axillary bromhidrosis.

A proteome scale study reveals how plastic surfaces and agitation promote protein aggregation.

Protein aggregation in biotherapeutics can reduce their activity and effectiveness. It may also promote immune reactions responsible for severe adverse effects. The impact of plastic materials on protein destabilization is not totally understood. Here, we propose to deconvolve the effects of material surface, air/liquid interface, and agitation to decipher their respective role in protein destabilization and aggregation. We analyzed the effect of polypropylene, TEFLON, glass and LOBIND surfaces on the stability of purified proteins (bovine serum albumin, hemoglobin and α-synuclein) and on a cell extract composed of 6000 soluble proteins during agitation (P = 0.1-1.2 W/kg). Proteomic analysis revealed that chaperonins, intrinsically disordered proteins and ribosomes were more sensitive to the combined effects of material surfaces and agitation while small metabolic oligomers could be protected in the same conditions. Protein loss observations coupled to Raman microscopy, dynamic light scattering and proteomic allowed us to propose a mechanistic model of protein destabilization by plastics. Our results suggest that protein loss is not primarily due to the nucleation of small aggregates in solution, but to the destabilization of proteins exposed to material surfaces and their subsequent aggregation at the sheared air/liquid interface, an effect that cannot be prevented by using LOBIND tubes. A guidance can be established on how to minimize these adverse effects. Remove one of the components of this combined stress - material, air (even partially), or agitation - and proteins will be preserved.

Lipoma management with a minimally invasive 1,444 nm Nd:YAG laser technique.

Background: Lipoma is the most common benign mesenchymal tumor that is composed of mature fat cells. Subdermal laser lipoma treatment may be recommended as an alternative to surgery for its removal. Purpose: The purpose of the study was to investigate the efficacy of the 1,444 nm Nd:YAG laser subcutaneous intralesional application as a treatment option for lipoma. Materials and methods: On 60 patients (37 women and 23 men) with lipomas localized above the muscle and lipomatosis in various regions, a subcutaneous, micro-pulsed 1,444 nm Nd:YAG laser procedure was executed. Before treatment, an ultrasound was performed and the lipomas were measured. The same lighting setup and photographic tools were used to take pictures of each patient. Results: The lipoma reduced or completely disappeared in all cases at the last follow-up, and no infections, burns, skin lesions, episodes of severe bleeding, or other serious adverse effects were reported. The most common transient side effects were ecchymosis and edema. Partial lesion reduction refers to rare cases of lipomatosis in which the lipomas were so small that suction and accurate positioning of the capsular membrane contours were impossible. Conclusion: Lipoma treatment with a 1,444 nm Nd:YAG laser is a safe and effective minimally invasive procedure without risk of scarring. For cellular disruption, laser treatment is an effective and safe option.

Synergistic Sequential Emission of Fractional 10.600 and 1540 nm Lasers for Skin Resurfacing: An Ex Vivo Histological Evaluation.

Background: Fractional ablative and non-ablative lasers are useful treatments for skin rejuvenation. A procedure that provides the sequential application of fractional ablative followed by non-ablative laser treatment may reduce patients' downtime and deliver better cosmetic results than with either laser alone. Objective: The purpose of the current study was to demonstrate the ameliorative and therapeutic effects in skin remodeling of the synergistic use of the two laser wavelengths (fractional ablative CO2 and non-ablative 1540 nm) with three different types of pulse shapes, S-Pulse (SP), D-Pulse (DP) and H-Pulse (HP), through which the CO2 laser can emit, performing an ex vivo histological evaluation. Methods: In this prospective study, ex vivo sheep inner thigh skin was chosen due to its similarity to human skin tissue, and a histological evaluation was performed. Three irradiation conditions, using all of the three CO2 pulse shapes (alone or averaged), were investigated: (1) 10.600 nm alone, the sequential irradiation of the two wavelengths in the same perfectly controlled energy pulses (DOT) for the entire scan area; ((2) 10.600 nm followed immediately by 1540 nm; and (3) 1540 nm followed immediately by 10.600 nm). Results: When comparing ablative to sequential irradiations, the synergy of the two wavelengths did not alter the typical ablative pulse shape of the 10.600 nm laser alone. With the same CO2 pulse shape, the lesion depth did not vary with the synergy of the two wavelengths, while thermal lesion width increased compared to CO2 alone. The ablation rate was achieved, while the total thermal lesion coverage in the scanning area of CO2 - 1540 lasers was greater than when using CO2 alone and then the other sequential irradiation. Conclusions: This study provides important preclinical data for new and early uses of the novel 10.600/1540 nm dual-wavelength non-ablative fractional laser. The synergy of the two wavelengths enhanced all the benefits already available when using CO2 laser systems both in terms of tone strengthening, thanks to a greater shrinking effect, and in terms of stimulation and collagen remodeling thanks to a greater volumetric thermal effect.

Skin Fractional Scar Treatment with a New Carbon Dioxide Scanner: Histological and Clinical Evaluation.

Background: The mechanism of action of fractional carbon dioxide (CO2) laser in the management of skin scarring is stimulation of collagen and fibroblasts in the dermis, resulting in remodeling and shrinking of the skin. Objective: The purpose of this research is to assess the safety and performance of a new CO2 laser scanner for treatment of acne scars. Methods: The study was carried out on 20 patients of both sexes, with a mean age of 25.7 ± 6.7 years. To assess the performance and safety of this new CO2 scanner, a preclinical histological evaluation was done. A clinical evaluation of acne scars was performed using Goodman and Baron's quantitative global acne scarring grading system (GBQGASGS) and a crusting scale where crusting scores ranged from 1 = none to 4 = severe. Digital photographs were taken to obtain esthetic results. Results: GBQGASGS showed a significant improvement in patients' scars and the treatment was well tolerated, with no lasting side effects. Conclusions: The new scanner seems to be an effective and safe device for skin scarring treatment, speeding up the healing time of scars.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Evaluation of safety and efficacy of a new device for muscle toning and body shaping.

BACKGROUND: The spread of non-invasive procedures for fat deposits removal has increased rapidly in recent years. In the field of esthetic medicine, high-intensity focused electromagnetic field (HIFEM) technology has recently been introduced, as a tool for toning and strengthening muscles, which goes far beyond normal physical exercise. OBJECTIVE: The purpose of this study is to evaluate the efficacy and safety of a new device for body remodeling. METHODS: A set of 15 patients (7 males and 8 females, BMI 24.05 ± 2.01 kg m-2 , age 32-57) participated in this study. Patients were enrolled at Dermatos center, Montesilvano, Abruzzo, Italy. The technology used is FMS (Flat Magnetic Stimulation): 6-8 treatment sessions were performed. The sessions must be repeated twice a week, with a minimum of 2 days between each session. Treatment duration varies from 20 to 45 min, depending on patients. RESULTS: During 1-month follow-up after the last treatment evaluations, the results showed tonification, the strengthening of muscles, and the reduction of localized adiposity. There is a significant reduction in waist circumference (80.7 ± 4.3 cm vs 77.3 ± 5.6 cm, p < 0.001). All patients showed relatively high satisfaction immediately after the last treatment. CONCLUSIONS: Our data show that intense muscle activity is generated by FMS treatments, suggesting that this technology could be used as a convenient and effective muscle toning tool.

Could 675-nm Laser Treatment Be Effective for Facial Melasma Even in Darker Phototype?

Objective: The study assesses the safety and efficacy of a 675-nm laser source on melasma. Background: Melasma is a frequent acquired skin disease defined by the presence of hyperpigmented macules on the face. Methods: Study protocol included up to three sessions of the 675-nm laser. Objective evaluation was assessed by using a 5-point visual analogue pain scale (VAS) (range, 0-4). Treatment tolerance was assessed using the 5-point VAS. Results: A mean 3.1 ± 0.7 improvement was reached according to photographic evaluation by VAS. Patients treated one time showed mean clearance of 3.3 ± 0.76, patients treated two times showed mean clearance of 3.0 ± 0.71, and patients treated three times showed mean clearance of 3.0 ± 1. Pain score mean was 1.2 ± 0.4. Conclusions: The 675-nm laser system seems to be safe and effective even in the treatment of facial melasma in patients with Fitzpatrick phototypes IV to V.

Characterization and Ex Vivo Application of Indocyanine Green Chitosan Patches in Dura Mater Laser Bonding.

Dura mater repair represents a final and crucial step in neurosurgery: an inadequate dural reconstruction determines dreadful consequences that significantly increase morbidity and mortality rates. Different dural substitutes have been used with suboptimal results. To overcome this issue, in previous studies, we proposed a laser-based approach to the bonding of porcine dura mater, evidencing the feasibility of the laser-assisted procedure. In this work, we present the optimization of this approach in ex vivo experiments performed on porcine dura mater. An 810-nm continuous-wave AlGaAs (Aluminium Gallium Arsenide) diode laser was used for welding Indocyanine Green-loaded patches (ICG patches) to the dura. The ICG-loaded patches were fabricated using chitosan, a resistant, pliable and stable in the physiological environment biopolymer; moreover, their absorption peak was very close to the laser emission wavelength. Histology, thermal imaging and leak pressure tests were used to evaluate the bonding effect. We demonstrated that the application of 3 watts (W), pulsed mode (Ton 30 ms, Toff 3.5 ms) laser light induces optimal welding of the ICG patch to the dura mater, ensuring an average fluid leakage pressure of 216 ± 105 mmHg, falling within the range of physiological parameters. This study demonstrated that the thermal effect is limited and spatially confined and that the laser bonding procedure can be used to close the dura mater. Our results showed the effectiveness of this approach and encourage further experiments in in vivo models.