Clathrin-mediated endocytosis in budding yeast at a glance.

Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process.

Primary Cutaneous Interdigitating Dendritic Cell Sarcoma: A Case Report and Review of the Literature.

Interdigitating dendritic cell sarcoma (IDCS) is defined as a neoplastic proliferation of spindle to ovoid cells with phenotypic features similar to those of interdigitating dendritic cells, which are present in the T cell-rich areas of lymphoid organs and participate as antigen-presenting cells responsible for initiating primary T lymphocyte immune response. IDCS usually presents with lymphadenopathy. Solitary lymph node involvement is often seen. Extra nodal presentation has been described as well. Cutaneous lesions are extremely rare, and less than 10 cases have been previously documented in medical literature. Here, the authors describe another primary cutaneous IDCS in a 42-year-old patient and review the literature.

The use of the 300 microsecond 1064 nm Nd:YAG laser in the treatment of keloids.

BACKGROUND: Keloids can be quite resistant to conventional methods of treatment. A wide range of treatment modalities exists, often with suboptimal results, recurrences, and adverse events occurring. Laser therapy with the carbon dioxide, erbium:YAG, Q switched frequency doubled neodymium-doped yttrium aluminium garnet (Nd:YAG), and 585/595 nm pulsed dye lasers have all be purported as potential treatment modalities however with limited efficacy and data especially in the skin of color population is limited. We report the successful use of the 300 microsecond 1064 nm Nd:YAG laser in treating keloids in patients with skin types ranging from Fitzpatrick I through VI with special attention in treating skin of color patients. OBJECTIVE: We examined the use of the 300 microsecond 1064 nanometer (nm) Nd:YAG laser for the treatment keloids in patients with skin types ranging from Fitzpatrick I through VI. METHODS & MATERIALS: A retrospective analysis of treatment efficacy was conducted on 44 patients with keloids. Three separate treatment groups were compared. The groups consisted of: a "control group" in which the whole keloid was only treated with intralesional corticosteroid (triamcinolone 10 mg/cc) (16 patients); a "laser only" group in which the patient's keloid was only treated with the 1064 nm Nd:YAG laser at a fluency of 13 to 18 Joules / centimeter2 (J/cm2), a fixed pulse duration of 300 microseconds, 5 mm spot size, and a total of 2000 pulses (14 patients); and a "combination group" that received both the aforementioned laser therapy and adjuvant intralesional triamcinolone (14 patients). RESULTS: Patients in the "combination group" treated with the 300 microsecond 1064 nm Nd:YAG laser therapy plus intralesional corticosteroid and the "laser only" group both were observed to have durable clinical reduction in the thickness and erythema of the keloids. These results were shown to be superior to the "control group" whom were only treated with intralesional corticosteroids. Only mild and transient post treatment erythema was noted as an adverse effect. STATISTICAL ANALYSIS: Data analysis was performed using IBM SPSS Statistics 19.0.0 (Armonk, NY). In order to assess the statistical significance of differences in keloid improvement among the three treatment groups, The Kruskal-Wallis test (non-parametric ANOVA test) was applied. The level of statistical significance was set at P< 0.05. A statistically significant difference in keloid improvement was appreciated between treatment groups (P<0.0001). LIMITATIONS: A small sample size and the retrospective nature of the analysis are limitations to the study.
CONCLUSION: The 300 microsecond 1064 nm Nd:YAG laser proved effective in improving the clinical appearance of keloids. We recommended this laser protocol in conjunction with intralesional corticosteroids as a treatment option for patients with keloids, especially in the skin of color population. The 1064 nm Nd:YAG laser did not show post inflammatory hyperpigmentation nor hypopigmenatation, which are concerns for skin types IV to VI, and therefore is a suitable option for such patients.

Management of Ocular Toxicity in Patients Receiving Belantamab Mafodotin.

While significant strides have been made in the treatment of multiple myeloma, treatment options remain limited and definite, and most patients ultimately succumb to their disease. The urgency for more treatment modalities remains, as patients who are refractory to proteasome inhibitors, immunomodulatory agents, and anti-CD38 monoclonal antibodies have a median survival of only 5.8 to 13 months. Belantamab mafodotin, a first-in-class antibody-drug conjugate, was approved by the US Food and Drug Administration in 2020 for patients with relapsed or refractory myeloma who have received at least four prior therapies, including an anti-CD38 monoclonal antibody, a proteasome inhibitor, and an immunomodulatory agent. It produced an overall response rate of 31%, and the median progression-free survival was 2.9 months when administered as a single agent. While generally well tolerated, ocular toxicities were a notable adverse event reported. In this article, we discuss the response data, toxicity profile including ocular toxicities, and treatment management.

A Focus on Waldenström Macroglobulinemia and AL Amyloidosis.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma that is characterized by the overproduction of an IgM monoclonal protein. It may cause adenopathy, hepatomegaly, splenomegaly, as well as other disease-related complications such as cold agglutinin anemia, cryoglobulinemia, hyperviscosity, and neuropathy. While light chain amyloidosis in patients with WM only occurs in about 10% of patients, it is important that advanced practitioners are able to recognize concurrent AL amyloidosis, which will affect the patient's treatment trajectory. Diagnosis of WM with AL amyloidosis is based on bone marrow biopsy and a fat pad biopsy. If AL amyloidosis is suspected, the bone marrow and fat pad biopsy should undergo Congo red staining. If it is negative, and there is a strong suspicion of AL amyloidosis, then an organ biopsy can be considered. Treatment of WM uses rituximab-based therapy in combination with a variety of other agents, including proteasome inhibitors, alkylating agents, and BTK inhibitors. Treatment of light chain amyloidosis uses bortezomib as the backbone of therapy and can be administered with cyclophosphamide, dexamethasone, and now daratumumab, which was recently approved. Waldenström macroglobulinemia and light chain amyloidosis are both rare diseases and can lead to a variety of disease-related complications. Fortunately, many options exist for both diseases. This article will highlight a case of WM with amyloidosis and a case of a patient with relapsing WM with considerations for advanced practitioners managing this patient population.

Human immunodeficiency virus-1 Tat activates calpain proteases via the ryanodine receptor to enhance surface dopamine transporter levels and increase transporter-specific uptake and Vmax.

Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, "reversible" DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson's disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously, we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here, we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT V(max), without changes in K(m) or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3ß. We conclude that Tat activates RyRs via a calcium- and calpain-mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3ß inhibition as clinical therapeutic approaches for HAND. Finally, we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.

AL Amyloidosis: Unfolding a Complex Disease.

Light chain (AL) amyloidosis is a rare plasma cell dyscrasia. An estimated 12,000 people live with the disease in the United States. AL amyloidosis occurs from the misfolding of proteins that deposit in organs (heart, kidneys, digestive tract, tongue, lungs, and nervous system), leading to progressive organ damage and impairment of quality of life. The treatment of AL amyloidosis has improved greatly over the past several years, with new treatments currently in development. This article will focus on the pathophysiology, diagnosis, and treatment of AL amyloidosis.

Selection and stabilization of endocytic sites by Ede1, a yeast functional homologue of human Eps15.

During clathrin-mediated endocytosis (CME), endocytic-site maturation can be divided into two stages corresponding to the arrival of the early and late proteins at the plasma membrane. The early proteins are required to capture cargo and position the late machinery, which includes proteins involved in actin assembly and membrane scission. However, the mechanism by which early-arriving proteins select and stabilize endocytic sites is not known. Ede1, one of the earliest proteins recruited to endocytic sites, facilitates site initiation and stabilization. Deletion of EDE1 results in fewer CME initiations and defects in the timing of vesicle maturation. Here we made truncation mutants of Ede1 to better understand how different domains contribute to its recruitment to CME sites, site selection, and site maturation. We found that the minimal domains required for efficient Ede1 localization at CME sites are the third EH domain, the proline-rich region, and the coiled-coil region. We also found that many strains expressing ede1 truncations could support a normal rate of site initiation but still had defects in site-maturation timing, indicating separation of Ede1 functions. When expressed in yeast, human Eps15 localized to the plasma membrane, where it recruited late-phase CME proteins and supported productive endocytosis, identifying it as an Ede1 functional homologue.

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis.

Actin polymerization powers membrane deformation during many processes, including clathrin-mediated endocytosis (CME). During CME in yeast, actin polymerization is triggered and coordinated by a six-protein WASP/Myosin complex that includes WASP, class I myosins (Myo3 and Myo5), WIP (Vrp1), and two other proteins. We show that a single engineered protein can replace this entire complex while still supporting CME. This engineered protein reveals that the WASP/Myosin complex has four essential activities: recruitment to endocytic sites, anchorage to the plasma membrane, Arp2/3 activation, and transient actin filament binding by the motor domain. The requirement for both membrane and F-actin binding reveals that myosin-mediated coupling between actin filaments and the base of endocytic sites is essential for allowing actin polymerization to drive membrane invagination.

Casein kinase 1 promotes initiation of clathrin-mediated endocytosis.

In budding yeast, over 60 proteins functioning in at least five modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization.

The ansacarbamitocins: polar ansamitocin derivatives.

The ansacarbamitocins are a new family of maytansinoids that are unusually substituted with a glucose subunit and two carbamate functional groups and exhibit modest activity against some agricultural fungal disease organisms. Ansacarbamitocins A-F ( 1- 6) all consist of the same macrocyclic core as the ansamitocins, with variation occurring on the glucose unit, while ansacarbamitocins A1 and B1 ( 7, 8) additionally lack the epoxide group on C-4 and C-5.