Off and On Again: De- and Reubiquitination during Membrane Protein Degradation.

In this issue of Molecular Cell, Hu et al. (2020) show that the cytosolic E3 ligase RNF126 reubiquitinates membrane proteins after their extraction from the membrane of the endoplasmic reticulum to target them for proteasomal degradation.

Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling.

Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.

Prolonged inhibition of intratumoral mast cells enhances efficacy of low-dose antiangiogenic therapy.

Low-dose antiangiogenic therapies have demonstrated the ability to enhance normalization of tumor vessels, consequently improving hypoxia levels, drug delivery, and promoting anticancer immune responses. Mast cells have been identified as contributors to resistance against antiangiogenic therapy and facilitators of abnormal neoangiogenesis. In this study, we demonstrate that by simultaneously targeting intratumoral mast cells with Imatinib and administering low-dose anti-VEGFR2 therapy, antitumor efficacy can be enhanced in preclinical models. Thus, combinatory treatment overcomes therapy resistance, while concurrently promoting tumor vessel normalization. Notably, histomorphometric analysis of tumor sections revealed that vessel perfusion could be improved through mast cell inhibition and, despite a significantly reduced microvessel density, the combination treatment did not result in elevated tumor hypoxia levels compared to anti-VEGFR2 therapy alone. Short-term Imatinib application effectively increased antitumor efficacy, and by prolonging the application of Imatinib tumor vessel normalization was additionally improved. The combination of mast cell depletion and antiangiogenic treatments has not been investigated in detail and promises to help overcoming therapy resistance. Further studies will be required to explore their impact on other treatment approaches, and subsequently to validate these findings in a clinical setting.

PET Imaging of Self-Assembled 18 F-Labelled Pd2 L4 Metallacages for Anticancer Drug Delivery.

To advance the design of self-assembled metallosupramolecular architectures as new generation theranostic agents, the synthesis of 18 F-labelled [Pd2 L4 ]4+ metallacages is reported. Different spectroscopic and bio-analytical methods support the formation of the host-guest cage-cisplatin complex. The biodistribution profiles of one of the cages, alone or encapsulating cisplatin have been studied by PET/CT imaging in healthy mice in vivo, in combination to ICP-MS ex vivo.

Synthesis of 177Lu-Labeled, Somatostatin-2 Receptor-Targeted Metalla-Assemblies: Challenges in the Design of Supramolecular Radiotherapeutics.

Self-assembled supramolecular coordination complexes (SCCs) hold promise for biomedical applications in cancer therapy, although their potential in the field of nuclear medicine is still substantially unexplored. Therefore, in this study an exo-functionalized cationic [Pd2L2]4+ metallacycle (L = 3,5-bis(3-ethynylpyridine)phenyl), targeted to the somatostatin-2 receptor (sst2R) and featuring the DOTA chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) in order to bind the ß-- and γ-emitter lutetium-177, was synthesized by self-assembly following ligand synthesis via standard solid-phase peptide synthesis (SPPS). This metallacycle was then characterized by reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI-MS), and 1H and 1H-DOSY NMR (DOSY = diffusion-ordered spectroscopy). A procedure for the radiolabeling of the metallacycle with 177Lu was also optimized. The resulting [nat/177Lu]Lu-DOTA-metallacycle, termed [nat/177Lu]Lu-Cy, was evaluated concerning its stability and in vitro properties. The compound was more lipophilic compared to the reference [177Lu]Lu-DOTA-TATE (logPOct/H2O = -0.85 ± 0.10 versus -3.67 ± 0.04, respectively). While [natLu]Lu-Cy revealed low stability in a DMEM/F12 GlutaMax medium, it demonstrated good stability in other aqueous media as well as in DMSO. A high sst2R binding affinity (expressed as IC50) was determined in CHOsst2 cells (Chinese hamster ovary cells that were stably transfected with human sst2R). Moreover, the metallacycle exhibited high human serum albumin binding, as assessed by high-performance affinity chromatography (HPAC), and moderate stability in human serum compared to [177Lu]Lu-DOTA-TATE (TATE = (Tyr3)-octreotate). In order to improve stability, a heteroleptic approach was used to develop a less sterically hindered cage-like SCC that is potentially endowed with host-guest chemistry capability, which has been preliminarily characterized by RP-HPLC and ESI-MS. Overall, our initial results encourage future studies on sst2R-directed SCCs and have led to new insights into the chemistry of ss2R-directed SCCs for radiopharmaceutical applications.

The Effect of Renal Denervation on T Cells in Patients with Resistant Hypertension.

(1) Background: Sympathetic overactivity is a major contributor to resistant hypertension (RH). According to animal studies, sympathetic overactivity increases immune responses, thereby aggravating hypertension and cardiovascular outcomes. Renal denervation (RDN) reduces sympathetic nerve activity in RH. Here, we investigate the effect of RDN on T-cell signatures in RH. (2) Methods: Systemic inflammation and T-cell subsets were analyzed in 17 healthy individuals and 30 patients with RH at baseline and 6 months after RDN. (3) Results: The patients with RH demonstrated higher levels of pro-inflammatory cytokines and higher frequencies of CD4+ effector memory (TEM), CD4+ effector memory residential (TEMRA) and CD8+ central memory (TCM) cells than the controls. After RDN, systolic automated office blood pressure (BP) decreased by -17.6 ± 18.9 mmHg. Greater BP reductions were associated with higher CD4+ TEM (r -0.421, p = 0.02) and CD8+ TCM (r -0.424, p = 0.02) frequencies at baseline. The RDN responders, that is, the patients with ≥10mmHg systolic BP reduction, showed reduced pro-inflammatory cytokine levels, whereas the non-responders had unchanged inflammatory activity and higher CD8+ TEMRA frequencies with increased cellular cytokine production. (4) Conclusions: The pro-inflammatory state of patients with RH is characterized by altered T-cell signatures, especially in non-responders. A detailed analysis of T cells might be useful in selecting patients for RDN.

Oral Anticancer Heterobimetallic PtIV -AuI Complexes Show High In Vivo Activity and Low Toxicity.

AuI -carbene and PtIV -AuI -carbene prodrugs display low to sub-µM activity against several cancer cell lines and overcome cisplatin (cisPt) resistance. Linking a cisPt-derived PtIV (phenylbutyrate) complex to a AuI -phenylimidazolylidene complex 2, yielded the most potent prodrug. While in vivo tests against Lewis Lung Carcinoma showed that the prodrug PtIV (phenylbutyrate)-AuI -carbene (7) and the 1 : 1 : 1 co-administration of cisPt: phenylbutyrate:2 efficiently inhibited tumor growth (≈95 %), much better than 2 (75 %) or cisPt (84 %), 7 exhibited only 5 % body weight loss compared to 14 % for 2, 20 % for cisPt and >30 % for the co-administration. 7 was much more efficient than 2 at inhibiting TrxR activity in the isolated enzyme, in cells and in the tumor, even though it was much less efficient than 2 at binding to selenocysteine peptides modeling the active site of TrxR. Organ distribution and laser-ablation (LA)-ICP-TOFMS imaging suggest that 7 arrives intact at the tumor and is activated there.

Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Neuropsychological Evidence for a Motor Working Memory Subsystem Related to Apraxia.

Recent evidence in healthy participants suggests that a motor subcomponent of working memory (mWM) may exist. We investigated whether this mWM is impaired in patients with a motor-dominant left hemisphere (LH) stroke and apraxia. Furthermore, we hypothesized that a deficient mWM contributes to deficits in motor cognition, that is, apraxia, in LH stroke. The study included 52 patients with LH stroke and 25 age-matched controls. Patients were classified into LH stroke patients with and without apraxia based on deficits in gesture imitation and object use. All participants were examined using the block span test (visuospatial WM), the digit span test (verbal WM), and a novel mWM task. In the latter, participants were presented with static pictures depicting three different actions: actions with objects, meaningless actions, and meaningful actions. In the mWM task, LH stroke patients with apraxia performed worse than age-matched controls. Notably, LH stroke patients with apraxia showed more pronounced mWM deficits than those without apraxia. These results remained significant even after controlling for visuospatial and verbal WM deficits. Regression analyses revealed that LH stroke patients' mWM deficits predicted deficits in imitation. Data provide neuropsychological evidence for a motor subsystem of WM and suggest that deficits in mWM contribute to the severity of apraxia in LH stroke patients.

Intensity of mycophenolate mofetil treatment is associated with an impaired immune response to SARS-CoV-2 vaccination in kidney transplant recipients.

Kidney transplant recipients (KTRs) are extremely vulnerable to SARS-CoV-2 infection and show an impaired immune response to SARS-CoV-2 vaccination. We analyzed factors related to vaccination efficiency in KTRs. In a multicenter prospective observational study (NCT04743947), IgG antibodies levels against SARS-CoV-2 spike S1 subunit and their neutralization capacity after SARS-CoV-2 vaccination were analyzed in 225 KTRs and compared to 176 controls. After the vaccination, 56 (24.9%) KTRs became seropositive of whom 68% had neutralizing antibodies. This immune response was significantly lower compared to controls (239 [78-519] BAU/ml versus 1826 [560-3180] BAU/ml for KTRs and controls, p < .0001). The strongest predictor for an impaired response was mycophenolate mofetil (MMF) treatment. Multivariate regression analysis revealed that MMF-free regimen was highly associated with seroconversion (OR 13.25, 95% CI 3.22-54.6; p < .001). In contrast, other immunosuppressive drugs had no significant influence. 187 out of 225 KTRs were treated with MMF of whom 26 (13.9%) developed antibodies. 23 of these seropositive KTRs had a daily MMF dose ≤1 g. Furthermore, higher trough MMF concentrations correlated with lower antibody titers (R -0.354, p < .001) supporting a dose-dependent unfavorable effect of MMF. Our data indicate that MMF dose modification could lead to an improved immune response.

Immune response to third SARS-CoV-2 vaccination in seronegative kidney transplant recipients: Possible improvement by mycophenolate mofetil reduction.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) µg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

"Dynamical Docking" of Cyclic Dinuclear Au(I) Bis-N-heterocyclic Complexes Facilitates Their Binding to G-Quadruplexes.

With the aim to improve the design of metal complexes as stabilizers of noncanonical DNA secondary structures, namely, G-quadruplexes (G4s), a series of cyclic dinuclear Au(I) N-heterocyclic carbene complexes based on xanthine and benzimidazole ligands has been synthesized and characterized by various methods, including X-ray diffraction. Fluorescence resonance energy transfer (FRET) and CD DNA melting assays unraveled the compounds' stabilization properties toward G4s of different topologies of physiological relevance. Initial structure-activity relationships have been identified and recognize the family of xanthine derivatives as those more selective toward G4s versus duplex DNA. The binding modes and free-energy landscape of the most active xanthine derivative (featuring a propyl linker) with the promoter sequence cKIT1 have been studied by metadynamics. The atomistic simulations evidenced that the Au(I) compound interacts noncovalently with the top G4 tetrad. The theoretical results on the Au(I) complex/DNA Gibbs free energy of binding were experimentally validated by FRET DNA melting assays. The compounds have also been tested for their antiproliferative properties in human cancer cells in vitro, showing generally moderate activity. This study provides further insights into the biological activity of Au(I) organometallics acting via noncovalent interactions and underlines their promise for tunable targeted applications by appropriate chemical modifications.

The Ubiquitin-Specific Protease Usp7, a Novel Merkel Cell Polyomavirus Large T-Antigen Interaction Partner, Modulates Viral DNA Replication.

Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA.IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.

Bioconjugate Supramolecular Pd2+ Metallacages Penetrate the Blood Brain Barrier In Vitro and In Vivo.

The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- ⊂ CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- ⊂ cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.

Ruthenium-based PACT agents based on bisquinoline chelates: synthesis, photochemistry, and cytotoxicity.

The known ruthenium complex [Ru(tpy)(bpy)(Hmte)](PF6)2 ([1](PF6)2, where tpy = 2,2':6',2″-terpyridine, bpy = 2,2'-bipyridine, Hmte = 2-(methylthio)ethanol) is photosubstitutionally active but non-toxic to cancer cells even upon light irradiation. In this work, the two analogs complexes [Ru(tpy)(NN)(Hmte)](PF6)2, where NN = 3,3'-biisoquinoline (i-biq, [2](PF6)2) and di(isoquinolin-3-yl)amine (i-Hdiqa, [3](PF6)2), were synthesized and their photochemistry and phototoxicity evaluated to assess their suitability as photoactivated chemotherapy (PACT) agents. The increase of the aromatic surface of [2](PF6)2 and [3](PF6)2, compared to [1](PF6)2, leads to higher lipophilicity and higher cellular uptake for the former complexes. Such improved uptake is directly correlated to the cytotoxicity of these compounds in the dark: while [2](PF6)2 and [3](PF6)2 showed low EC50 values in human cancer cells, [1](PF6)2 is not cytotoxic due to poor cellular uptake. While stable in the dark, all complexes substituted the protecting thioether ligand upon light irradiation (520 nm), with the highest photosubstitution quantum yield found for [3](PF6)2 (Φ[3] = 0.070). Compounds [2](PF6)2 and [3](PF6)2 were found both more cytotoxic after light activation than in the dark, with a photo index of 4. Considering the very low singlet oxygen quantum yields of these compounds, and the lack of cytotoxicity of the photoreleased Hmte thioether ligand, it can be concluded that the toxicity observed after light activation is due to the photoreleased aqua complexes [Ru(tpy)(NN)(OH2)]2+, and thus that [2](PF6)2 and [3](PF6)2 are promising PACT candidates.

Thermostable water reservoirs in the interlayer space of a sodium hectorite clay through the intercalation of γ-aminopropyl(dimethyl)ethoxysilane in toluene.

Treatment of Na+-based hectorite LAPONITE® (LAP) and of Na+-montmorillonite (MMT) with a homologous series of γ-aminopropyl(methyl)x(ethoxy)ysilanes (x + y = 3, y > 0) in toluene was studied by means of thermogravimetric analysis coupled with mass spectrometry, infrared spectroscopy, 29Si and 23Na solid-state nuclear magnetic resonance spectroscopy and powder X-ray diffraction. The triethoxy silane (APTS) exclusively grafts on the clays' edges as branched oligomers whereas both the monoethoxy silane (APMS) and the diethoxy silane (APDS) are also intercalated, the latter as linear oligomers. Intercalation of APMS varies for MMT and LAP: MMT hosts the smallest amounts of the silanes with marginal increase of the basal distance and no stabilization of water. On the contrary, LAP accommodates the largest amount of guests in the form of monomeric APMS which yields the largest increase of the basal distance and stabilizes water up to 200 °C when APMS dimerizes. APMS stabilization is attributed to intramolecular Si-O-H-NH2 hydrogen bonds and the hydrophobic geminal methyl groups together with the trimethylene sides of the cyclic monomers are thought to compartmentalize the hydrated sodium sites. The high temperature release of water from APMS@LAP is discussed in the light of potentially triggered interphase degradation in composite materials for recycling purposes.

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

Ru(III) Complexes with Lonidamine-Modified Ligands.

A series of bifunctional Ru(III) complexes with lonidamine-modified ligands (lonidamine is a selective inhibitor of aerobic glycolysis in cancer cells) was described. Redox properties of Ru(III) complexes were characterized by cyclic voltammetry. An easy reduction suggested a perspective for these agents as their whole mechanism of action seems to be based on activation by metal atom reduction. New compounds demonstrated a more pronounced antiproliferative potency than the parental drug; individual new agents were more cytotoxic than cisplatin. Stability studies showed an increase in the stability of complexes along with the linker length. A similar trend was noted for antiproliferative activity, cellular uptake, apoptosis induction, and thioredoxin reductase inhibition. Finally, at concentrations that did not alter water solubility, the selected new complex evoked no acute toxicity in Balb/c mice.

Multiaction Pt(IV) Carbamate Complexes Can Codeliver Pt(II) Drugs and Amine Containing Bioactive Molecules.

Multiaction Pt(IV) prodrugs can overcome resistance associated with the FDA approved Pt(II) drugs like cisplatin. Intracellular reduction of the octahedral Pt(IV) derivatives of cisplatin releases cisplatin and the two axial ligands. When the released axial ligands act synergistically with cisplatin to kill the cancer cells, we have multiaction prodrugs. Most Pt(IV) multiaction prodrugs have bioactive ligands possessing a carboxylate that is conjugated to the Pt(IV) because breaking the Pt(IV)-ligand bond releases the active moiety. As many drugs that act synergistically with cisplatin do not have carboxylates, a major challenge is to prepare multiaction Pt(IV) complexes with drugs that have amino groups or hydroxyl groups such that following reduction, the drugs are released in their active form. Our objective was to prepare multiaction Pt(IV) prodrugs that release bioactive molecules having amino groups. Because we cannot conjugate amino groups to the axial position of Pt(IV), we developed a novel and efficient approach for the synthesis of Pt(IV)-carbamato complexes and demonstrated that following reduction of the Pt(IV), the released carbamates undergo rapid decarboxylation, releasing the free amine, as in the case of the PARP-1 inhibitor 3-aminobenzamide and the amino derivative of the HDAC inhibitor SAHA. Pt(IV)-carbamato complexes are stable in cell culture medium and are reduced by ascorbate. They are reduced slower than their carboxylato and carbonato analogues. We believe that this approach paves the way for preparing novel classes of multiaction Pt(IV) prodrugs with amino containing bioactive molecules that up to now were not accessible.

[Child abuse-reduction in the estimated number of unreported cases by restructuring a clinical child protection program]. / Kindeswohlgefährdung ­ Ausweitung des Hellfeldes durch Neukonzeption der klinischen Kinderschutzarbeit.

BACKGROUND: Identification of child abuse is a daily challenge in medical work. The estimated number of unreported cases of child abuse and neglect is high. OBJECTIVES: The aim of this study was to investigate the effectiveness of the redesigned clinical child protection program of a major German pediatric hospital and to improve programs in other hospitals for children and physicians through presentation of the advantages of the new structure. METHODS: All cases of child protection at the Altona Children's Hospital were retrospectively analyzed before and after restructuring of the clinic's child protection program for a two-year period each, and a comparison was made. The child protection program was restructured and the new program subsequently managed by a fulltime coordinator. RESULTS: The prevalence of both suspected and substantiated cases of child abuse was significantly higher after restructuring of the child protection program. Before the change, 24 cases were investigated, of which 23 were substantiated; afterward, 124 cases were investigated and 89 were substantiated. Despite the high number of false-positive suspected cases, stigmatization of the families during the clarification progress was avoided by using a very sensitive approach, and the family was not confronted until the suspicion was affirmed. CONCLUSION: The presented concept of child protection in medical clinics seems to facilitate a higher detection rate of child abuse cases. The reduction in the number of undetected cases and thereby prevention of possible escalation of abuse, as well as the decline in the immense social follow-up costs, justifies the increased personnel costs.