Evolutionary analysis identifies a Golgi pathway and correlates lineage-specific factors with endomembrane organelle emergence in apicomplexans.

The organelle paralogy hypothesis (OPH) aims to explain the evolution of non-endosymbiotically derived organelles. It predicts that lineage-specific pathways or organelles should result when identity-encoding membrane-trafficking components duplicate and co-evolve. Here, we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in Apicomplexa, a globally important parasitic lineage. We are able to identify 18 paralogs of known membrane-trafficking machinery, in several cases co-incident with the presence of new endomembrane organelles in apicomplexans or their parent lineage, the Alveolata. Moreover, focused analysis of the apicomplexan Arf-like small GTPases (i.e., ArlX3) revealed a specific post-Golgi trafficking pathway. This pathway appears involved in delivery of proteins to micronemes and rhoptries, with knockdown demonstrating reduced invasion capacity. Overall, our data have identified an unforeseen post-Golgi trafficking pathway in apicomplexans and are consistent with the OPH mechanism acting to produce endomembrane pathways or organelles at various evolutionary stages across the alveolate lineage.

Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites.

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii.

Comparative Life Cycle Assessment Between Single-Use and Reprocessed IPC Sleeves.

Purpose: Healthcare has a large environmental footprint, not least due to the wide use of single-use supplies. Reprocessing of medical devices is a well-established, regulated process, and can reduce its environmental impact. This life cycle assessment (LCA) compares the environmental footprint of a single-use and a reprocessed version of otherwise identical intermittent pneumatic compression (IPC) sleeves. Materials and Methods: The LCA was performed in accordance with the international standard ISO 14044 using the Environmental Footprint 3.0 (EF) method for the assessment. Data were obtained in cooperation with IPC sleeve manufacturers. Where no primary data were available, ecoinvent database records were used. The functional unit is five hospital treatments applying IPC. The robustness of the results was interrogated in sensitivity analyses of the energy mix, the ethylene oxide emissions during reprocessing, and the transport distances. The impact of waste reduction on hospital disposal costs was calculated. Results: The environmental footprint of reprocessed IPC sleeves was found to be reduced in all categories compared to single-use devices, leading to a weighted normalized reduction of 43% across all categories. In a breakdown of the LCA results, reprocessed IPC sleeves were found to reduce the carbon footprint by 40%, with the treatment of five patients with single-use IPC sleeves creating 7 kg CO2eq, compared to 4.2 kg CO2eq from reprocessed sleeves. Waste disposal costs were also reduced by 90%. Conclusion: Reprocessing of IPC sleeves provides an environmental and economic benefit in comparison to single-use devices.

The SUN-like protein TgSLP1 is essential for nuclear division in the apicomplexan parasite Toxoplasma gondii.

Connections between the nucleus and the cytoskeleton are important for positioning and division of the nucleus. In most eukaryotes, the linker of nucleoskeleton and cytoskeleton (LINC) complex spans the outer and inner nuclear membranes and connects the nucleus to the cytoskeleton. In opisthokonts, it is composed of Klarsicht, ANC-1 and Syne homology (KASH) domain proteins and Sad1 and UNC-84 (SUN) domain proteins. Given that the nucleus is positioned at the posterior pole of Toxoplasma gondii, we speculated that apicomplexan parasites must have a similar mechanism that integrates the nucleus and the cytoskeleton. Here, we identified three UNC family proteins in the genome of the apicomplexan parasite T. gondii. Whereas the UNC-50 protein TgUNC1 localised to the Golgi and appeared to be not essential for the parasite, the SUN domain protein TgSLP2 showed a diffuse pattern throughout the parasite. The second SUN domain protein, TgSLP1, was expressed in a cell cycle-dependent manner and was localised close to the mitotic spindle and, more detailed, at the kinetochore. We demonstrate that conditional knockout of TgSLP1 leads to failure of nuclear division and loss of centrocone integrity.

Evaluating the Environmental Impact of Single-Use and Multi-Use Surgical Staplers with Staple Line Buttressing in Laparoscopic Bariatric Surgery.

Purpose: Operation rooms have a large environmental impact. Single-use staplers (SUS) are widely used surgical instruments that contribute to resource consumption and waste generation, whereas multi-use staplers (MUS) can greatly reduce the environmental impact of surgery. The staple lines are often reinforced with buttressing material to prevent leaks and bleeding. We explore current clinical practice and environmental concerns regarding stapling and buttressing, as well as the environmental impact of staple line buttressing in sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). Furthermore, we extend this analysis by taking packaging material and the lithium in power supplies into consideration. Materials and Methods: A survey of bariatric surgeons was conducted to assess stapler and buttressing use in clinical practice. We deconstructed and analyzed the product and packaging composition of a commonly used SUS with separate staple line reinforcement (Echelon Flex™ with Echelon Endopath™, Ethicon) and MUS (Signia™ with Tri-Staple™ reinforced reloads, Medtronic), where the buttressing material was delivered separately or already incorporated in the reload cartridge, respectively. Both systems were compared regarding total waste generation, resource use (determined as total material requirement), and greenhouse gas emission caused by their lithium content. Results: 60 mm cartridges were most frequently used in bariatric surgery, and 67% of surveyed surgeons applied staple line reinforcement. MUS with pre-attached buttressing resulted in a reduction of waste, material consumption, and greenhouse gas emissions compared to SUS with separate buttressing: they reduced product waste by 40% (SG and RYBG), packaging waste by 60% (SG) and 57% (RYGB), resource consumption by more than 90%, and greenhouse gas emissions related to the lithium in the batteries by 99.7%. Preloaded buttressing produced less waste than separate buttressing per stapler firing. Conclusion: The environmental impact of surgery can be greatly reduced by using MUS with pre-attached buttressing rather than SUS with separate buttressing.

Liver-directed treatment is associated with improved survival and increased response to immune checkpoint blockade in metastatic uveal melanoma: results from a retrospective multicenter trial.

Metastases of uveal melanoma (UM) spread predominantly to the liver. Due to low response rates to systemic therapies, liver-directed therapies (LDT) are commonly used for tumor control. The impact of LDT on the response to systemic treatment is unknown. A total of 182 patients with metastatic UM treated with immune checkpoint blockade (ICB) were included in this analysis. Patients were recruited from prospective skin cancer centers and the German national skin cancer registry (ADOReg) of the German Dermatologic Cooperative Oncology Group (DeCOG). Two cohorts were compared: patients with LDT (cohort A, n = 78) versus those without LDT (cohort B, n = 104). Data were analyzed for response to treatment, progression-free survival (PFS), and overall survival (OS). The median OS was significantly longer in cohort A than in cohort B (20.1 vs. 13.8 months; P = 0.0016) and a trend towards improved PFS was observed for cohort A (3.0 vs. 2.5 months; P = 0.054). The objective response rate to any ICB (16.7% vs. 3.8%, P = 0.0073) and combined ICB (14.1% vs. 4.5%, P = 0.017) was more favorable in cohort A. Our data suggest that the combination of LDT with ICB may be associated with a survival benefit and higher treatment response to ICB in patients with metastatic UM.

Customized 3D-printed molds for high dose-rate brachytherapy in facial skin cancer: First clinical experience.

BACKGROUND AND OBJECTIVE: Radiotherapy of elderly, frail patients with facial skin cancer in proximity to critical organs is challenging. This is the first report on clinical experience with facial skin cancer treated by individualized 3D-printer-based mold high-dose-rate (HDR) brachytherapy (BT). PATIENTS AND METHODS: Fifteen patients not eligible for radical surgery or definitive external beam radiotherapy (EBRT) were treated with 3D-printer-based mold HDR-BT. Patient selection and treatment were in accordance with multidisciplinary tumor board recommendations. Clinical response, toxicity and cosmesis were analyzed. RESULTS: Median age was 77 years. Histology revealed squamous cell carcinoma in seven, basal cell carcinoma in five, melanoma in situ in one, Lentigo maligna in one, and melanoma in one patient, respectively. Median prescription dose was 39 Gy delivered in once-daily fractions of 3 Gy. After a median follow-up of 12.2 months, local recurrence was observed in one patient with melanoma in situ. Apart from one grade 4 cataract, no other > grade 2 late toxicity was documented. CONCLUSIONS: HDR-BT with 3D-printer-based molds for facial skin cancer is a well-tolerated and safe treatment option for elderly, frail patients not eligible for radical surgery or definitive EBRT due to functional inoperability or tumor location.

A central CRMP complex essential for invasion in Toxoplasma gondii.

Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. During invasion, the unique secretory organelles (micronemes and rhoptries) are sequentially released and several micronemal proteins have been suggested to be required for host cell recognition and invasion. However, to date, only few micronemal proteins have been demonstrated to be essential for invasion, suggesting functional redundancy that might allow such a broad host range. Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan-specific proteins. In T. gondii, two CRMPs are present in the genome, CRMPA (TGGT1_261080) and CRMPB (TGGT1_292020). Here, we demonstrate that both proteins form a complex that contains the additional proteins MIC15 and the thrombospondin type 1 domain-containing protein (TSP1). Disruption of this complex results in a block of rhoptry secretion and parasites being unable to invade the host cell. In conclusion, this complex is a central invasion complex conserved in all apicomplexans.

Real-world outcomes using PD-1 antibodies and BRAF + MEK inhibitors for adjuvant melanoma treatment from 39 skin cancer centers in Germany, Austria and Switzerland.

BACKGROUND: Programmed death-1 (PD-1) antibodies and BRAF + MEK inhibitors are widely used for adjuvant therapy of fully resected high-risk melanoma. Little is known about treatment efficacy outside of phase III trials. This real-world study reports on clinical outcomes of modern adjuvant melanoma treatment in specialized skin cancer centers in Germany, Austria and Switzerland. METHODS: Multicenter, retrospective study investigating stage III-IV melanoma patients receiving adjuvant nivolumab (NIV), pembrolizumab (PEM) or dabrafenib + trametinib (D + T) between 1/2017 and 10/2021. The primary endpoint was 12-month recurrence-free survival (RFS). Further analyses included descriptive and correlative statistics, and a multivariate linear-regression machine learning model to assess the risk of early melanoma recurrence. RESULTS: In total, 1198 patients from 39 skin cancer centers from Germany, Austria and Switzerland were analysed. The vast majority received anti PD-1 therapies (n = 1003). Twelve-month RFS for anti PD-1 and BRAF + MEK inhibitor-treated patients were 78.1% and 86.5%, respectively (hazard ratio [HR] 1.998 [95% CI 1.335-2.991]; p = 0.001). There was no statistically significant difference in overall survival (OS) in anti PD-1 (95.8%) and BRAF + MEK inhibitor (96.9%) treated patients (p > 0.05) during the median follow-up of 17 months. Data indicates that anti PD-1 treated patients who develop immune-related adverse events (irAEs) have lower recurrence rates compared to patients with no irAEs (HR 0.578 [95% CI 0.443-0.754], p = 0.001). BRAF mutation status did not affect overall efficacy of anti PD-1 treatment (p > 0.05). In both, anti PD-1 and BRAF + MEK inhibitor treated cohorts, data did not show any difference in 12-month RFS and 12-month OS comparing patients receiving total lymph node dissection (TLND) versus sentinel lymph node biopsy only (p > 0.05). The recurrence prediction model reached high specificity but only low sensitivity with an AUC = 0.65. No new safety signals were detected. Overall, recorded numbers and severity of adverse events were lower than reported in pivotal phase III trials. CONCLUSIONS: Despite recent advances in adjuvant melanoma treatment, early recurrence remains a significant clinical challenge. This study shows that TLND does not reduce the risk of early melanoma recurrence and should only be considered in selected patients. Data further highlight that variables collected during clinical routine are unlikely to allow for a clinically relevant prediction of individual recurrence risk.

Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Functional Downregulation of PD-L1 and PD-L2 by CpG and non-CpG Oligonucleotides in Melanoma Cells.

The clinical application of immune checkpoint inhibitors represents a breakthrough progress in the treatment of metastasized melanoma and other tumor entities. In the present study, it was hypothesized that oligonucleotides (ODNs), known as modulators of the immune response, have an impact on the endogenous expression of checkpoint molecules, namely PD-L1 and PD-L2 (PD-L1/2). IFNγ-stimulated melanoma cells (A375, SK-Mel-28) were treated with different synthetically manufactured oligonucleotides which differed in sequence, length and backbone composition. It was found that a variety of different ODN sequences significantly suppressed PD-L1/2 expression. This effect was dependent on length and phosphorothioate (PTO) backbone. In particular, a sequence containing solely guanines (nCpG-6-PTO) was highly effective in downregulating PD-L1/2 at the protein, mRNA and promoter levels. Mechanistically, we gave evidence that ODNs with G-quartet-forming motifs suppress the interferon signaling axis (JAK/STAT/IRF1). Our findings identify a subset of ODNs as interesting pharmacological compounds that could expand the arsenal of targeted therapies to combat the immunological escape of tumor cells.

Factors Associated with Hemorrhage of Melanoma Brain Metastases after Stereotactic Radiosurgery in the Era of Targeted/Immune Checkpoint Inhibitor Therapies.

We aimed to evaluate the factors associated with hemorrhage (HA) of melanoma brain metastases (MBM) after Cyberknife stereotactic radiosurgery (SRS) in the modern era of systemic therapy. A total of 55 patients with 279 MBM were treated in 93 fractions. The median age, SRS dose, radiological follow-up, and time to HA were 60.4 years, 20 Gy, 17.7 months, and 10.7 months, respectively. Radiologically evident HA was documented in 47 (16.8%) metastases. Of the 55 patients, 25 (45.4%) suffered an HA. Among those, HA caused grade 3 toxicity in 10 patients (40%) and grade 1 symptoms in 5 patients (20%). Ten patients (40%) with HA experienced no toxicity. Logistic regression revealed the use of anticoagulants and the administration of systemic therapy within 7/15 days from SRS to be predictive for HA. When considering the HA causing grade 3 symptomatology, only the use of anticoagulants was significant, with the delivery of whole brain radiation therapy (WBRT) before the HA narrowly missing statistical significance. Our retrospective analysis showed that the administration of modern systemic therapy within 7/15 days from SRS may contribute to HA of MBM, though it appears safe, at least concerning grade 3 toxicity. The use of anticoagulants by the time of SRS significantly increased the risk of HA.

A splitCas9 phenotypic screen in Toxoplasma gondii identifies proteins involved in host cell egress and invasion.

Apicomplexan parasites, such as Toxoplasma gondii, have specific adaptations that enable invasion and exit from the host cell. Owing to the phylogenetic distance between apicomplexan parasites and model organisms, comparative genomics has limited capacity to infer gene functions. Further, although CRISPR/Cas9-based screens have assigned roles to some Toxoplasma genes, the functions of encoded proteins have proven difficult to assign. To overcome this problem, we devised a conditional Cas9-system in T. gondii that enables phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we screened 320 genes to identify those required for defined steps in the asexual life cycle. The detailed characterization of two genes identified in our screen, through the generation of conditional knockout parasites using the DiCre-system, revealed that signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, and a novel conoid protein (conoid gliding protein, CGP) functions late during egress and is required for the activation of gliding motility. Establishing different indicator lines and applying our conditional Cas9 screen could enable the identification of genes involved in organellar biogenesis, parasite replication or maintenance of the endosymbiotic organelles in the future.

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission.

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane-associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localizes at the cytoplasmic periphery and concavin(-) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(-) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(-) sporozoites are transmitted. Strikingly, motile concavin(-) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

Immune Checkpoint Blockade for Metastatic Uveal Melanoma: Re-Induction following Resistance or Toxicity.

Re-induction with immune checkpoint blockade (ICB) needs to be considered in many patients with uveal melanoma (UM) due to limited systemic treatment options. Here, we provide hitherto the first analysis of ICB re-induction in UM. A total of 177 patients with metastatic UM treated with ICB were included from German skin cancer centers and the German national skin cancer registry (ADOReg). To investigate the impact of ICB re-induction, two cohorts were compared: patients who received at least one ICB re-induction (cohort A, n = 52) versus those who received only one treatment line of ICB (cohort B, n = 125). In cohort A, a transient benefit of overall survival (OS) was observed at 6 and 12 months after the treatment start of ICB. There was no significant difference in OS between both groups (p = 0.1) with a median OS of 16.2 months (cohort A, 95% CI: 11.1-23.8) versus 9.4 months (cohort B, 95% CI: 6.1-14.9). Patients receiving re-induction of ICB (cohort A) had similar response rates compared to those receiving ICB once. Re-induction of ICB may yield a clinical benefit for a small subgroup of patients even after resistance or development of toxicities.

Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii.

Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites' ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.