Copper(II)-Catalyzed Amination of Aryl Chlorides in Aqueous Ammonia.

Anilines are ubiquitous in bio-active compounds and their synthesis can be achieved via metal-catalyzed cross-coupling reactions involving aryl halides. We describe an unusual, yet simple, CuII-catalyzed system for the amination of aryl chlorides in pure aqueous ammonia with 2.5 mol% catalyst loading under non-inert conditions. Different from previous systems, the reaction proceeds even without an additional organic solvent. Copper(II) sulfate in combination with 4,7-dimethoxy-1,10-phenanthroline enabled the amination of several aryl chlorides containing electron-neutral, -donating and -withdrawing groups to the corresponding anilines with good to excellent yields. The upscaling potential of the procedure has been shown by the synthesis at 50 mmol scale. The reaction proceeds as one of the rare cases of a CuII-assisted coupling, in contrast to the typical CuI-CuIII intermediates postulated for most Ullmann-type coupling reactions. The copper(II) center allows for a nucleophilic substitution pathway, enabled by the deprotonation of coordinated ammonia.

Virus production in shallow groundwater at the bank of the Danube River.

Viruses play a crucial role in regulating prokaryotic populations, yet their impact on subsurface environments, specifically groundwater habitats, remains poorly understood. In this study, we employed the virus-dilution approach to measure lytic virus production rates in shallow groundwater located near the city of Vienna (Austria) during the period from July-November 2020. Physico-chemical parameters (pH, electrical conductivity, water temperature, concentration of dissolved oxygen), prokaryotic, and viral abundance, and viral decay rates were monitored as well. Our findings revealed low virus-to-prokaryote ratios varying between 0.9-3.9 throughout the study period and a lack of correlation between prokaryotic and viral abundance in groundwater. Virus production rates varied between 9-12% of viral abundance h-1 in July-August and between 34-36% of viral abundance h-1 in October-November. Seasonal variations in virus production rates were found to be correlated with electrical conductivity, revealing ~3.5 times higher virus production rates during periods with high electrical conductivity and low groundwater recharge in October-November compared to July-August with higher groundwater recharge and lower electrical conductivity. Our data indicate that groundwater recharge disrupts the balance between virus and prokaryotic host communities, resulting in a deficiency of suitable prokaryotic host cells for viral proliferation.

Discordance of retroperitoneal and thoracic histologic findings in patients with metastatic germ cell tumors at postchemotherapy residual tumor resection.

INTRODUCTION AND OBJECTIVES: Postchemotherapy residual tumor resection (PC-RTR) is an important part of the multimodal treatment for patients with metastatic germ cell tumors. Simultaneous retroperitoneal and thoracic metastases often require consecutive surgical procedures. This study analyzes the histologic findings after abdominal and thoracic surgery in order to tailor the sequence and intensity of surgery. PATIENTS AND METHODS: From a total of 671 PC-RTRs from 2008 to 2021 we analyzed 50 patients with stage III non-seminomatous germ cell tumor (NSGCT) who had undergone both retroperitoneal and thoracic postchemotherapy residual tumor resection after first-line and salvage chemotherapy. RESULTS: All patients included had stage III NSGCT. 39 and 11 patients received first-line and salvage chemotherapy, respectively. 45 (90%) patients received retroperitoneal resection first, followed by thoracic surgery. Three patients (6%) underwent thoracic surgery before retroperitoneal surgery and two patients (4%) underwent simultaneous surgery. Overall, the histology of retroperitoneal and thoracic specimens was discordant in 23% of cases. After first-line chemotherapy, of fourteen patients with necrosis in retroperitoneal histology, four patients had vital carcinoma in lung histology. In patients with teratoma in the retroperitoneum, the thoracic findings were concordant in most cases (78%). When teratomatous elements were also present in the orchiectomy specimen, concordance was 100%. After salvage chemotherapy, the discordance rate was 55%. CONCLUSION: The data presented in this study underline that retroperitoneal residual masses with necrosis cannot reliably predict histologic findings of thoracic specimens. Patients with teratoma in the retroperitoneum have a high likelihood of teratoma in the thoracic specimen.

Engineering Mesoscale T Cell Receptor Clustering by Plug-and-Play Nanotools.

T cell receptor (TCR) clustering and formation of an immune synapse are crucial for TCR signaling. However, limited information is available about these dynamic assemblies and their connection to transmembrane signaling. In this work, TCR clustering is controlled via plug-and-play nanotools based on an engineered irreversible conjugation pair and a peptide-loaded major histocompatibility complex (pMHC) molecule to compare receptor assembly in a ligand (pMHC)-induced or ligand-independent manner. A streptavidin-binding peptide displayed in both tools enabled their anchoring in streptavidin-pre-structured matrices. Strikingly, pMHC-induced clustering in the confined regions exhibit higher density and dynamics than the ligand-free approach, indicating that the size and architecture of the pMHC ligand influences TCR assembly. This approach enables the control of membrane receptor clustering with high specificity and provides the possibility to explore different modalities of receptor activation.

Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Serological Evidence of Crimean-Congo Haemorrhagic Fever in Livestock in the Omaheke Region of Namibia.

This research examined the positivity ratio of Crimean-Congo haemorrhagic fever (CCHF) antibodies in cattle and sheep within Namibia's Omaheke region after a human disease outbreak in the same geographical area. A total of 200 samples (100 cattle and 100 sheep) were randomly collected from animals brought to two regional auction sites, and then tested using the ID Screen® CCHF Double Antigen Multi-Species Enzyme-Linked Immunosorbent Assay kit. Of the cattle samples, 36% tested positive, while 22% of the sheep samples were seropositive. The cattle had a significantly higher positivity ratio than sheep at the individual animal level (p = 0.0291). At the herd level, 62.5% of cattle herds and 45.5% of sheep flocks had at least one positive animal, but this difference was statistically insignificant (p = 0.2475). The fourteen cattle farms with at least one seropositive animal were dispersed across the Omaheke region. In contrast, the ten sheep farms with seropositive cases were predominantly situated in the southern half of the region. The study concluded that the CCHF is endemic in the Omaheke region and likely in most of Namibia, underscoring the importance of continued surveillance and preventive measures to mitigate the impact of CCHFV on animal health and potential spillover into human populations.

Cervical pre-cancer screening by visual inspection of the cervix after application of acetic acid in rural Burkina Faso: evaluation of women's knowledge, screening practice habits, acceptability and prevalence of risk factors and lesions in Boussé health district.

Introduction: cervical cancer is a major public health problem among women in sub-Saharan Africa. The disease can be controlled through early diagnosis through simple cost-effective methods such as visual inspection of the cervix after application of acetic acid or lugol´s iodine. However, screening for cervical cancer is still underused particularly in rural areas of Burkina Faso. The objective was to estimate the prevalence of cervical pre-cancer cancer in rural health district of Boussé, Burkina Faso. Methods: we conducted a cross-sectional study in the health district of Boussé in Northern-Central Burkina Faso from July to August 2014. Women aged 23-50 years were interviewed about their knowledge of cervical cancer and their screening practice and subsequently screened for cervical cancer by VIA. Results: a total of 418 participants were included with a median age of 34 years IQR (30-40 years). Two2 hundred participants (48%) had never heard about cervical cancer. About 134 participants (32%) knew at least one risk factor of cervical cancer. Only 37 women (9%) reported ever being screened for cervical cancer. Twenty-two percent reported concurrent sexual partnerships. The majority of the women (92%) are willing to pay to get screened for cervical pre-cancer by VIA. Overall, 21 participants (5%) were diagnosed with a cervical lesion by VIA and all of them accepted treatment with Loop electro surgical procedure. Conclusion: screening by VIA is feasible in rural Burkina Faso, but there is a poor knowledge on cervical cancer amongst the women. There is a need to set up a comprehensive, systematic, affordable and efficient cervical cancer program including an information campaign and making screening accessible in rural remote areas.

Drivers of microbial food-web structure along productivity gradients.

Ratios between viruses, heterotrophic prokaryotes and chlorophyll a are key indicators of microbial food structure and both virus-prokaryote and prokaryote-chlorophyll ratios have been proposed to decrease with system productivity. However, the mechanisms underlying these responses are still insufficiently resolved and their consistency across aquatic ecosystem types requires critical evaluation. We assessed microbial community ratios in highly productive African soda-lakes and used our data from naturally hypereutrophic systems which are largely underrepresented in literature, to complement earlier across-system meta-analyses. In contrast to marine and freshwater systems, prokaryote-chlorophyll ratios in African soda-lakes did not decrease along productivity gradients. High-resolution time series from two soda-lakes indicated that this lack of response could be driven by a weakened top-down control of heterotrophic prokaryotes. Our analysis of virus-prokaryote relationships, revealed a reduction of virus-prokaryote ratios by high suspended particle concentrations in soda-lakes. This effect, likely driven by the adsorption of free-living viruses, was also found in three out of four additionally analysed marine datasets. However, the decrease of virus-prokaryote ratios previously reported in highly productive marine systems, was neither detectable in soda-lakes nor freshwaters. Hence, our study demonstrates that system-specific analyses can reveal the diversity of mechanisms that structure microbial food-webs and shape their response to productivity increases.

The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I.

Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation.

Agrochemical Lessons for Infectious Disease Research: New Resistance Breaking Antifungal Hits against Candida auris.

Analysis of the history of the invention of the block-buster antifungal drug Fluconazole underscores the importance of agrochemical research on drug discovery and development. The multidrug resistant fungal pathogen Candida auris is now responsible for serious morbidity and mortality among immuno-compromised and long-term resident hospital patients globally. New drugs against C. auris are urgently needed. A focused screening of 1487 fungicides from the BASF agrochemical collection gave several potent inhibitors of C. auris with yet noncommercialized modes of action. The hits showed only minor activity loss against the azole-resistant C. auris strain CDC 0385 and low to moderate cytotoxicity to human HepG2 cells. Aminopyrimidine 4 showed high activity against resistant strains and selectivity in a HepG2 cells assay and is a potential hit candidate for further optimization.

Testicular germ cell tumours' clinical stage I: comparison of surveillance with adjuvant treatment strategies regarding recurrence rates and overall survival-a systematic review.

PURPOSE: Testicular germ cell tumours (GCTs) represent the most common malignancy in young adult males with two thirds of all cases presenting with clinical stage I (CSI). Active surveillance is the management modality mostly favoured by current guidelines. This systematic review assesses the treatment results in CSI patients concerning recurrence rate and overall survival in non-seminoma (NS) and pure seminoma (SE) resulting from surveillance in comparison to adjuvant strategies. METHODS/SYSTEMATIC REVIEW: We performed a systematic literature review confining the search to most recent studies published 2010-2021 that reported direct comparisons of surveillance to adjuvant management. We searched Medline and the Cochrane Library with additional hand-searching of reference lists to identify relevant studies. Data extraction and quality assessment of included studies were performed with stratification for histology (NS vs. SE) and treatment modalities. The results were tabulated and evaluated with descriptive statistical methods. RESULTS: Thirty-four studies met the inclusion criteria. In NS patients relapse rates were 12 to 37%, 0 to 10%, and 0 to 11.8% for surveillance, chemotherapy and for retroperitoneal lymph node dissection (RPLND) while overall survival rates were 90.7-100%, 91.7-100%, and 97-99.1%, respectively. In SE CSI, relapse rates were 0-22.3%, 0-5%, and 0-12.5% for surveillance, radiotherapy, chemotherapy, while overall survival rates were 84.1-98.7%, 83.5-100%, and 92.3-100%, respectively. CONCLUSION: In both histologic subgroups, active surveillance offers almost identical overall survival as adjuvant management strategies, however, at the expense of higher relapse rates. Each of the management strategies in CSI GCT patients have specific merits and shared-decision-making is advised to tailor treatment.

Structure of an MHC I-tapasin-ERp57 editing complex defines chaperone promiscuity.

Adaptive immunity depends on cell surface presentation of antigenic peptides by major histocompatibility complex class I (MHC I) molecules and on stringent ER quality control in the secretory pathway. The chaperone tapasin in conjunction with the oxidoreductase ERp57 is crucial for MHC I assembly and for shaping the epitope repertoire for high immunogenicity. However, how the tapasin-ERp57 complex engages MHC I clients has not yet been determined at atomic detail. Here, we present the 2.7-Å crystal structure of a tapasin-ERp57 heterodimer in complex with peptide-receptive MHC I. Our study unveils molecular details of client recognition by the multichaperone complex and highlights elements indispensable for peptide proofreading. The structure of this transient ER quality control complex provides the mechanistic basis for the selector function of tapasin and showcases how the numerous MHC I allomorphs are chaperoned during peptide loading and editing.

Semisynthetic Viral Inhibitor for Light Control of the MHC I Peptide Loading Complex.

The immune system detects virally or malignantly transformed cells via peptide-loaded major histocompatibility complex class I (pMHC I) molecules on the cell surface. MHC I molecules are loaded with cargo peptides in the endoplasmic reticulum (ER) by the highly dynamic multiprotein peptide loading complex (PLC). Here, we developed a semisynthetic approach to generate a photocleavable immune modulator ICP47 of Herpes simplex virus. Using this nanotool, we revealed key mechanistic events of the purified PLC, such as peptide binding and translocation coupled to ATP hydrolysis, triggered by light. We established a single-organelle flow cytometry assay to monitor light-controlled activation of the antigen processing machinery in native ER membranes. This photochemical modulation opens new opportunities for a comprehensive mechanistic analysis of the antigen processing machinery in vitro and native membrane environment.

Molecular basis of MHC I quality control in the peptide loading complex.

Major histocompatibility complex class I (MHC I) molecules are central to adaptive immunity. Their assembly, epitope selection, and antigen presentation are controlled by the MHC I glycan through a sophisticated network of chaperones and modifying enzymes. However, the mechanistic integration of the corresponding processes remains poorly understood. Here, we determine the multi-chaperone-client interaction network of the peptide loading complex (PLC) and report the PLC editing module structure by cryogenic electron microscopy at 3.7 Å resolution. Combined with epitope-proofreading studies of the PLC in near-native lipid environment, these data show that peptide-receptive MHC I molecules are stabilized by multivalent chaperone interactions including the calreticulin-engulfed mono-glucosylated MHC I glycan, which only becomes accessible for processing by α-glucosidase II upon loading of optimal epitopes. Our work reveals allosteric coupling between peptide-MHC I assembly and glycan processing. This inter-process communication defines the onset of an adaptive immune response and provides a prototypical example of the tightly coordinated events in endoplasmic reticulum quality control.

Prognostic factors in patients with clinical stage I nonseminoma-beyond lymphovascular invasion: a systematic review.

OBJECTIVE: To systematically evaluate evidence on prognostic factors for tumor recurrence in clinical stage I nonseminoma patients other than lymphovascular invasion (LVI). METHODS: We performed a systematic literature search in the biomedical databases Medline (via Ovid) and Cochrane Central Register of Controlled Trials (search period January 2010 to February 2021) for full text publications in English and German language, reporting on retro- or prospectively assessed prognostic factors for tumor recurrence in patients with stage I nonseminomatous germ cell tumors. RESULTS: Our literature search yielded eleven studies reporting on 20 potential prognostic factors. Results are based on cohort studies of mostly moderate to low quality. Five out of eight studies found a significant association of embryonal carcinoma (EC) in the primary tumor with relapse. Among the different risk definitions of embryonal carcinoma (presence, predominance, pure), presence of EC alone seems to be sufficient for prognostification. Interesting results were found for rete testis invasion, predominant yolk sac tumor, T-stage and history of cryptorchidism, but the sparse data situation does not justify their clinical use. CONCLUSIONS: No additional factors that meet the prognostic value of LVI, especially when determined by immunohistochemistry, could be identified through our systematic search. The presence of EC might serve as a second, subordinate prognostic factor for clinical use as the data situation is less abundant than the one of LVI. Further efforts are necessary to optimize the use of these two prognostic factors and to evaluate and validate further potential factors with promising preliminary data.

How to classify, diagnose, treat and follow-up extragonadal germ cell tumors? A systematic review of available evidence.

PURPOSE: To present the current evidence and the development of studies in recent years on the management of extragonadal germ cell tumors (EGCT). METHODS: A systematic literature search was conducted in Medline and the Cochrane Library. Studies within the search period (January 2010 to February 2021) that addressed the classification, diagnosis, prognosis, treatment, and follow-up of extragonadal tumors were included. Risk of bias was assessed and relevant data were extracted in evidence tables. RESULTS: The systematic search identified nine studies. Germ cell tumors (GCT) arise predominantly from within the testis, but about 5% of the tumors are primarily located extragonadal. EGCT are localized primarily mediastinal or retroperitoneal in the midline of the body. EGCT patients are classified according to the IGCCCG classification. Consecutively, all mediastinal non-seminomatous EGCT patients belong to the "poor prognosis" group. In contrast mediastinal seminoma and both retroperitoneal seminoma and non-seminoma patients seem to have a similar prognosis as patients with gonadal GCTs and metastasis at theses respective sites. The standard chemotherapy regimen for patients with a EGCT consists of 3-4 cycles (good vs intermediate prognosis) of bleomycin, etoposid, cisplatin (BEP); however, due to their very poor prognosis patients with non-seminomatous mediastinal GCT should receive a dose-intensified or high-dose chemotherapy approach upfront on an individual basis and should thus be referred to expert centers Ifosfamide may be exchanged for bleomycin in cases of additional pulmonary metastasis due to subsequently planned resections. In general patients with non-seminomatous EGCT, residual tumor resection (RTR) should be performed after chemotherapy. CONCLUSION: In general, non-seminomatous EGCT have a poorer prognosis compared to testicular GCT, while seminomatous EGGCT seem to have a similar prognosis to patients with metastatic testicular seminoma. The current insights on EGCT are limited, since all data are mainly based on case series and studies with small patient numbers and non-comparative studies. In general, systemic treatment should be performed like in testicular metastatic GCTs but upfront dose intensification of chemotherapy should be considered for mediastinal non-seminoma patients. Thus, EGCT should be referred to interdisciplinary centers with utmost experience in the treatment of germ cell tumors.

First-line salvage treatment options for germ cell tumor patients failing stage-adapted primary treatment : A comprehensive review compiled by the German Testicular Cancer Study Group.

PURPOSE: In this review, we summarize and discuss contemporary treatment standards and possible selection criteria for decision making after failure of adjuvant or first-line cisplatin-based chemotherapy for primarily localized or metastatic germ cell tumors. METHODS: This work is based on a systematic literature search conducted for the elaboration of the first German clinical practice guideline to identify prospective clinical trials and retrospective comparative studies published between Jan 2010 and Feb 2021. Study end points of interest were progression-free (PFS) and overall survival (OS), relapse rate (RR), and/or safety. RESULTS: Relapses of clinical stage I (CS I) patients irrespective of prior adjuvant treatment after orchiectomy are treated stage adapted in accordance for primary metastatic patients. Surgical approaches for sole retroperitoneal relapses are investigated in ongoing clinical trials. The appropriate salvage chemotherapy for metastatic patients progressing or relapsing after first-line cisplatin-based chemotherapy is still a matter of controversy. Conventional cisplatin-based chemotherapy is the international guideline-endorsed standard of care, but based on retrospective data high-dose chemotherapy and subsequent autologous stem cell transplantation may offer a 10-15% survival benefit for all patients. Secondary complete surgical resection of all visible residual masses irrespective of size is paramount for treatment success. CONCLUSIONS: Patients relapsing after definite treatment of locoregional disease are to be treated by stage-adapted first-line standard therapy for metastatic disease. Patients with primary advanced/metastatic disease failing one line of cisplatin-based combination chemotherapy should be referred to GCT expert centers. Dose intensity is a matter of ongoing debate, but sequential high-dose chemotherapy seems to improve patients' survival.

Can magnetic resonance imaging replace conventional computerized tomography for follow-up of patients with testicular cancer? A systematic review.

PURPOSE: Follow-up protocols for patients with testicular cancer (TC) have significantly reduced the number of cross-sectional imaging studies to reduce radiation exposure. At present, it is unclear whether magnetic resonance imaging (MRI) could replace conventional computerized tomography (CT) imaging. The objective of this study is to summarize the scientific evidence on this topic and to review guideline recommendations with regard to the use of MRI. METHODS: A systematic literature review was performed searching Medline and Cochrane databases for prospective studies on patients with TC in the follow-up care (last search in February 2021). Additionally, guideline recommendations for TC were screened. Data extraction and quality assessment of included studies were performed and used for a descriptive presentation of results. RESULTS: A total of four studies including two ongoing trials were identified. Overall, the scientific evidence of prospective comparative studies is based on 102 patients. Data suggest that abdominal imaging with MRI can replace conventional CT for detection of lymph node metastasis of the retroperitoneum to spare radiation exposure and contrast media application. However, experienced radiologists are needed. Clinical guidelines are aware of the risk of diagnosis-induced secondary malignancy due to CT imaging and some have adapted their recommendations accordingly. Results of the two ongoing trials on 738 patients are expected soon to provide more reliable results on this topic. CONCLUSIONS: There is growing evidence that abdominopelvic MRI imaging can replace CT imaging during follow-up of patients with TC in order to reduce radiation exposure and diagnosis-induced secondary malignancy.

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies.

Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

Do we need a more flexible use of Team Timeout calling? Evidence from the Handball Bundesliga.

Calling a Team Timeout (TTO) is one of the coaches' most important tools. Given the key competitive advantage to determine your own timing, it is crucial to make a good decision when to use a TTO. Existing research shows that teams can benefit in general from TTOs and that they are called at the end of the game and when trailing (Gomes et al., 2014; Gutiérrez-Aguilar et al., 2016; Prieto et al., 2016). However, to generate relevant findings, situational variables must be included (Fernandez-Navarro et al., 2020; Gómez, Lago-Peñas et al., 2015). By integrating situational variables like scoring streak and player difference and higher-order interactions, this study aims to identify specific game situations where TTOs are most effective. Based on 850 games of the German Handball Bundesliga, game situations are identified by Classification Tree Analysis and efficacies are evaluated. Findings indicate a strong impact of timing. Frequently used TTOs, e.g., at the end of periods, are beneficial to the teams. However, strongest effect occurs for TTOs taken at the early stages of the game and with a positive run. Results indicate that TTO is a powerful tactical tool and an application at uncommon timings may even enhance the success rate.