Longitudinal assessment of Leydig cell function in male survivors of childhood cancer.

BACKGROUND: As the number and longevity of childhood cancer survivors increases, assessing treatment-associated late effects remains crucial. We longitudinally examined the incidence of and associated risk factors for Leydig cell dysfunction (LCD) and Leydig cell failure (LCF) in men treated for pediatric cancers at our institution. PROCEDURE: We performed a retrospective longitudinal cohort study of adult male survivors treated for various childhood cancers who are at risk for LCD. The outcomes of interest were serum testosterone and luteinizing hormone (LH) levels during childhood and adulthood. Risk factors assessed included treatment with stem cell transplant, total body irradiation (TBI), and exposure to alkylating agents. RESULTS: Out of 118 eligible subjects, 7.6% had LCF and 14.4% had LCD. Median age at last testosterone level was 20 years. Subjects with sufficient testosterone levels in adulthood (N = 105) remained sufficient for a mean of 11.1 years following completion of cancer treatment. We found significant associations between LCF and treatment with TBI (p < .003) and between LCF in adulthood and testosterone insufficiency in childhood (p < .001). No statistically significant association was found between LCF and cyclophosphamide equivalent dose greater than 20 g/m2 (p = .2). LCF/LCD occurred in a small number of nonirradiated patients treated with the highest doses of alkylators. CONCLUSIONS: Incidence of LCF and LCD are low in male survivors of childhood cancer. Longitudinally, there is an association between childhood testosterone insufficiency and LCF in adulthood. Alkylating agents and stem cell transplant without TBI were not associated with LCF in our study.

Single-Molecule Force Spectroscopy of Deoxyribonucleic Acid and Deoxyribonucleic Acid Polymerase Activity Impacted by Alkylating Agents.

DNA alkylating agents are widely used in anticancer pharmacology. Although shown to induce cross-linking and/or methylation of DNA, how they affect the mechanical properties of DNA and activity of DNA enzymes remains to be elucidated. Here, we perform single-molecule optical tweezer experiments on DNA treated with alkylating agents, including melphalan, cisplatin, and dacarbazine. While all three drugs induce a significant increase of overstretching force and a reduction of hysteresis, suggesting stabilization of DNA against shearing forces, their effects on elasticity of DNA were quite different, with the largest change in persistence length induced by cisplatin. Furthermore, we find that these alkylating-agent-induced changes on DNA have different effects on processivity of DNA polymerase, with melphalan and cisplatin showing significantly reduced activity and dacarbazine showing little effect. Overall, our results provide new insights into the effects for these alkylating agents, which could potentially facilitate a better design of related drugs.

DNA Alkylation Damage by Nitrosamines and Relevant DNA Repair Pathways.

Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts.

Synthesis and antiproliferative activities of steroidal lactam conjugates bearing a new nitrogen mustard.

Alkylating agents are potent anticancer compounds that exert their anticancer properties through the inhibition of cell replication and transcription leading to cell death. Despite the numerous benefits, these agents also have serious drawbacks such as their high toxicity and low specificity towards cancer cells. As previously reported by our group, conjugation of alkylating agents with azasteroids can reduce their systemic toxicity and enhance their anticancer activity. In this work, novel steroidal alkylating agents bearing POPAM-OH were synthesized and their anticancer efficacy was evaluated in vitro and in vivo. All the novel hybrids demonstrated high antiproliferative effects against 5 different cancer cell lines in the low micromolar range. Treatment of SCID mice bearing SKOV-3 or PC-3 tumor xenografts with the most potent hybrid 19 led to significant reduction of tumor size (tumor inhibition TI = 95% in SKOV3 models and TI = 85.2% in PC3 models). Importantly, the acute toxicity of hybrid 19 (LD10 = 36 µΜ, LD50 = 62 µΜ) in CB17 SCID mice exhibited three-fold decrease compared to the acute toxicity of previously reported hybrids of POPAM-NH2. This is an important finding since systemic cytotoxicity is a critical limitation of alkylating agents. Collectively, the steroidal conjugates of POPAM-OH displayed significant anticancer efficacy and reduced toxicity in vitro and in vivo rendering them as good candidates for cancer therapy.

Electrochemistry of chemotherapeutic alkylating agents and their interaction with DNA.

Alkylating agents were among the first anticancer drugs to be discovered and continue to be the most commonly used in chemotherapy. They are electrophiles that react with the ring nitrogen and extracyclic oxygen atoms of DNA bases, forming covalent adducts that further lead to cross-linking of DNA strands, abnormal base pairing or DNA strand breaks. The investigation and quantitative analysis of alkylating agents in biological samples are essential for monitoring the therapy progression and efficiency, understanding their pharmacokinetics and develop new more effective and specific chemotherapeutical drugs. Among biotechnological methods, electrochemical techniques are particularly important in pharmaceutical medicine, owing to their rapid detection, great sensitivity, robustness, exceptional detection limits, ability to be used with small analyte volumes in turbid biofluids, and easy adaptability to miniaturization and point-of-care (POC) testing. This article provides first an exhaustive review concerning the electrochemical methods of characterization and quantification of different classes of chemotherapeutic alkylating agents (triazenes and hydrazines, nitrosoureas, nitrogen mustards, oxazaphosphorines, alkyl alkane sulfonates and ethylene imines) in standard samples, pharmaceutical formulations and biological matrixes. The second part of the article focuses on the recent electrochemical methodologies and DNA-electrochemical biosensors developed to study the interaction of alkylating agents with DNA. These studies are relevant for obtaining real-time details about the alkylating agents' mechanism of action and for assessing the oxidative DNA damage they cause, important for the development of improved antineoplastic drugs.

When to cryopreserve ovarian tissue: Determining the effects of chemotherapy on the ovarian reserve by studying follicular density and apoptosis.

OBJECTIVES: Patients scheduled to receive chemotherapy should be counselled on fertility preservation. Known gonadotoxic chemotherapies such as alkylating agents have a high risk of altering ovarian reserve. In some cases, the urgency of treatment requires the use of chemotherapy before fertility preservation, which will be carried out at a later stage. Usually the ovarian tissue is cryopreserved. The aim of our study is to investigate the impact of chemotherapies on follicular density and the apoptosis of reserve follicles. METHODS: We included 140 patients: 63 patients, mean age 18.8 years, were included in the group "no chemotherapy" (group A) and 77 patients, mean age 17.1 years, in the group "received chemotherapy before ovarian conservation" (group B). None of the patients had had pelvic radiotherapy prior to ovarian cryopreservation. The histological parameters studied were follicular density and the presence of malignant cells. We selected 12 patients from group A and 15 patients from group B, comparable in age and pathology, for whom we evaluated follicle apoptosis by immunostaining cleaved caspase-3. RESULTS: We demonstrated an inverse relationship between follicular density and age (p < 0.0001), as well as a lack of effect of chemotherapy on follicular density (p = 0.87). We showed the impact of various chemotherapies, especially with alkylating agents, on the apoptosis of ovarian follicles (p < 0.0001). Three patients had ovarian tissue infiltration, two of which were malignant. CONCLUSION: This work underlines the fact that conservation of ovarian tissue after chemotherapy remains possible.

Smurf1 Suppression Enhances Temozolomide Chemosensitivity in Glioblastoma by Facilitating PTEN Nuclear Translocation.

The tumor suppressor PTEN mainly inhibits the PI3K/Akt pathway in the cytoplasm and maintains DNA stability in the nucleus. The status of PTEN remains therapeutic effectiveness for chemoresistance of the DNA alkylating agent temozolomide (TMZ) in glioblastoma (GB). However, the underlying mechanisms of PTEN's interconnected role in the cytoplasm and nucleus in TMZ resistance are still unclear. In this study, we report that TMZ-induced PTEN nuclear import depends on PTEN ubiquitylation modification by Smurf1. The Smurf1 suppression decreases the TMZ-induced PTEN nuclear translocation and enhances the DNA damage. In addition, Smurf1 degrades cytoplasmic PTEN K289E (the nuclear-import-deficient PTEN mutant) to activate the PI3K/Akt pathway under TMZ treatment. Altogether, Smurf1 interconnectedly promotes PTEN nuclear function (DNA repair) and cytoplasmic function (activation of PI3K/Akt pathway) to resist TMZ. These results provide a proof-of-concept demonstration for a potential strategy to overcome the TMZ resistance in PTEN wild-type GB patients by targeting Smurf1.

Biosynthesis of DNA-Alkylating Antitumor Natural Products.

DNA-alkylating natural products play an important role in drug development due to their significant antitumor activities. They usually show high affinity with DNA through different mechanisms with the aid of their unique scaffold and highly active functional groups. Therefore, the biosynthesis of these natural products has been extensively studied, especially the construction of their pharmacophores. Meanwhile, their producing strains have evolved corresponding self-resistance strategies to protect themselves. To further promote the functional characterization of their biosynthetic pathways and lay the foundation for the discovery and rational design of DNA alkylating agents, we summarize herein the progress of research into DNA-alkylating antitumor natural products, including their biosynthesis, modes of action, and auto-resistance mechanisms.

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

The UVSSA protein is part of a genome integrity homeostasis network with links to transcription-coupled DNA repair and ATM signaling.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

MPTAC links alkylation damage signaling to sterol biosynthesis.

Overproduction of reactive oxygen species (ROS) drives inflammation and mutagenesis. However, the role of the DNA damage response in immune responses remains largely unknown. Here we found that stabilization of the mismatch repair (MMR) protein MSH6 in response to alkylation damage requires interactions with the molybdopterin synthase associating complex (MPTAC) and Ada2a-containing histone acetyltransferase complex (ATAC). Furthermore, MSH6 promotes sterol biosynthesis via the mevalonate pathway in a MPTAC- and ATAC-dependent manner. MPTAC reduces the source of alkylating agents (ROS). Therefore, the association between MMR proteins, MPTAC, and ATAC promotes anti-inflammation response and reduces alkylating agents. The inflammatory responses measured by xanthine oxidase activity are elevated in Lymphoblastoid Cell Lines (LCLs) from some Fragile X-associated disorders (FXD) patients, suggesting that alkylating agents are increased in these FXD patients. However, MPTAC is disrupted in LCLs from some FXD patients. In LCLs from other FXD patients, interaction between MSH6 and ATAC was lost, destabilizing MSH6. Thus, impairment of MPTAC and ATAC may cause alkylation damage resistance in some FXD patients.

Relevant pharmacological interactions between alkylating agents and antiepileptic drugs: Preclinical and clinical data.

Seizures are relatively common in cancer patients, and co-administration of chemotherapeutic and antiepileptic drugs (AEDs) is highly probable and necessary in many cases. Nonetheless, clinically relevant interactions between chemotherapeutic drugs and AEDs are rarely summarized and pharmacologically described. These interactions can cause insufficient tumor and seizure control or lead to unforeseen toxicity. This review focused on pharmacokinetic and pharmacodynamic interactions between alkylating agents and AEDs, helping readers to make a rational choice of treatment optimization, and thus improving patients' quality of life. As an example, phenobarbital, phenytoin, and carbamazepine, by increasing the hepatic metabolism of cyclophosphamide, ifosfamide and busulfan, yield smaller peak concentrations and a reduced area under the plasma concentration-time curve (AUC) of the prodrugs; alongside, the maximum concentration and AUC of their active products were increased with the possible onset of severe adverse drug reactions. On the other side, valproic acid, acting as histone deacetylase inhibitor, showed synergistic effects with temozolomide when tested in glioblastoma. The present review is aimed at providing evidence that may offer useful suggestions for rational pharmacological strategies in patients with seizures symptoms undertaking alkylating agents. Firstly, clinicians should avoid the use of enzyme-inducing AEDs in combination with alkylating agents and prefer the use of AEDs, such as levetiracetam, that have a low or no impact on hepatic metabolism. Secondly, a careful therapeutic drug monitoring of both alkylating agents and AEDs (and their active metabolites) is necessary to maintain therapeutic ranges and to avoid serious adverse reactions.

Alkylating Agents, the Road Less Traversed, Changing Anticancer Therapy.

Cancer is considered one of the gruelling challenges and poses a grave health hazard across the globe. According to the International Agency for Research on Cancer (IARC), new cancer cases increased to 18.1 million in 2018, with 9.6 million deaths, bringing the global cancer rate to 23.6 million by 2030. In 1942, the discovery of nitrogen mustard as an alkylating agent was a tremendous breakthrough in cancer chemotherapy. It acts by binding to the DNA, and creating cross linkages between the two strands, leading to halt of DNA replication and eventual cell death. Nitrogen lone pairs of 'nitrogen mustard' produce an intermediate 'aziridinium ion' at the molecular level, which is very reactive towards DNA of tumour cells, resulting in multiple side effects with therapeutic consequences. Owing to its high reactivity and peripheral cytotoxicity, several improvements have been made with structural modifications for the past 75 years to enhance its efficacy and improve the direct transport of drugs to the tumour cells. Alkylating agents were among the first non-hormonal substances proven to be active against malignant cells and also the most valuable cytotoxic therapies available for the treatment of leukaemia and lymphoma patients. This review focus on the versatile use of alkylating agents and the Structure Activity Relationship (SAR) of each class of these compounds. This could provide an understanding for design and synthesis of new alkylating agents having enhanced target specificity and adequate bioavailability.

Repeated Low-Dose Acrolein Triggers Irreversible Lamina Propria Edema in Urinary Bladder, Transient Voiding Behavior and Widening of Eyes to Mechanical Stimuli.

Acrolein is a metabolite of cyclophosphamide (CYP), an alkylating agent used for a wide range of benign and malignant diseases. CYP treatments are known to trigger hemorrhagic cystitis in patients and animals. Significant effort has been made to prevent CYP/acrolein-induced cystitis, while still maintaining its therapeutic benefits. As a result, supplementary therapeutic options to mediate the protective role against CYP/acrolein and lower doses of CYP are currently given to targeted patients, as compared to past treatments. There is still a need to further study the effects of the repeated low-dose CYP/acrolein on the pathophysiology of the urinary bladder. In our study, a one-time treatment of acrolein and repeated low-dose acrolein triggered the thickening of the smooth muscle and lamina propria in the urinary bladder of C57BL/6J mice, respectively. The first dose of acrolein did not trigger voiding dysfunction, but the second dose triggered high-volume low-frequency voiding. Interestingly, our new scoring criteria and concurrent behavioral assessment revealed that mice with repeated low-dose acrolein had a wider opening of eyes in response to mechanical stimuli. Our study suggests that clinical symptoms among patients undergoing prolonged low-dose CYP may differ from previously reported symptoms of CYP-induced hemorrhagic cystitis.

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase.

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway for Ku eviction. Here, we identify the pre-mRNA splicing protein XAB2 as a factor required for resistance to seDSBs induced by the chemotherapeutic alkylator temozolomide. Moreover, we show that XAB2 prevents Ku retention and abortive HR at seDSBs induced by temozolomide and camptothecin, via a pathway that operates in parallel to the ATM-CtIP-MRE11 axis. Although XAB2 depletion preserved RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased NHEJ engagement in S/G2 and genetic instability. Overexpression of RAD51 or RAD52 rescued the XAB2 defects and XAB2 loss was synthetically lethal with RAD52 inhibition, providing potential perspectives in cancer therapy.

Assessment of SENP3-interacting proteins in hepatocytes treated with diethylnitrosamine by BioID assay.

SUMOylation of proteins regulates cell behaviors and is reversibly removed by small ubiquitin-like modifier (SUMO)-specific proteases (SENPs). The SENP family member SENP3 is involved in SUMO2/3 deconjugation and has been reported to sense cell stress and accumulate in several human cancer cells and macrophages. We previously reported that Senp3-knockout heterozygous mice showed smaller liver, but the pertinent mechanisms of SENP3 and SUMOylated substrates remain unclear. Thus, in this study, we investigated the interacting proteins with SENP3 and the alteration in hepatocytes treated with the xenobiotic diethylnitrosamine (DEN), which is specifically transformed in the liver and induces DNA double-strand breaks. Our data revealed that a certain amount of SENP3 was present in normal, untreated hepatocytes; however, DEN treatment promoted rapid SENP3 accumulation. SENP3 was mainly localized in the nuclei, and its level was significantly increased in the cytoplasm after 2 h of DEN treatment. The application of the recent proximity-dependent biotinylation (BioID) method led to the identification of 310 SENP3-interacting proteins that were involved in not only gene transcription but also RNA splicing, protein folding, and metabolism. Furthermore, after DEN exposure for a short duration, ribosomal proteins as well as proteins associated with mitochondrial ATP synthesis, membrane transport, and bile acid synthesis, rather than DNA repair proteins, were identified. This study provides insights into the diverse regulatory roles of SENP3, and the BioID method seems to be efficient for identifying physiologically relevant insoluble proteins.

Effects of busulfan on somatic cells after inhibiting germ cells in the gonads of the young olive flounder Paralichthys olivaceus.

Busulfan is widely used in some species to inhibit germ cell proliferation. This study was conducted to evaluate effects of busulfan on germ and somatic cells in gonads of olive flounder, Paralichthys olivaceus, one of the most economically important mariculture fish species. After intraperitoneal injection with 80 (80B) or 120 (120B) mg/kg busulfan, both gonads were atrophied, and ovaries were discolored with adhesion to the visceral mass. Histological results indicated that germ cells in the gonads were detached, and there was a larger nucleus size and smaller cytoplasmic volume in spermatogonia. Numbers of oocytes and somatic cells in the ovary were both less (P < 0.05), while in the testis, numbers of spermatogonia and somatic cells were markedly lesser and greater, respectively (P < 0.05). In ovaries of the flounder treated with 80B and 120B, relative abundance of vasa and cyp19a1a mRNA transcripts was very small in the cytoplasm of oocytes, while the cyp19a1a transcript was still present in theca cells. In the testis of flounder treated with 80B and 120B, abundance of vasa was markedly less (P < 0.05) with there being very little vasa in spermatogonia and disruption of the spermatogonium structure. In the 80B treatment group, amh was in lesser abundance with there being very little amh in spermatogonia, however, with the 120B treatment there was a large amh abundance in spermatogonium with there being disruption of structure of these germ cells and Sertoli cells. Busulfan, therefore, might inhibit the development of spermatogonia in the flounder testis.

In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models.

Most transgenic animals are generated using a genome-modified stem cell system and genome modification directly in embryos. Although this system is well-established in the development of transgenic animals, donor cell-derived transgenic animal production is inefficient in some cases. Especially in avian models such as chickens, the efficiency of transgenic animal production through primordial germ cells (PGCs) is highly variable compared with embryonic manipulation of mammalian species. Because germ cell and germline-competent stem cell-mediated systems that contain the transgene are enriched only at the upstream level during cell cultivation, the efficiency of transgenic animal production is unreliable. Therefore, we developed an in vivo selection model to enhance the efficiency of transgenic chicken production using microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to the alkylating agent busulfan, which induces germ cell-specific cytotoxicity. Under in vitro conditions, MGSTII-tg PGCs were resistant to 1 µM busulfan, which was highly toxic to wild-type PGCs. In germline chimeric roosters, transgene-expressing germ cells were dominantly colonized in the recipient testes after busulfan exposure compared with non-treated germline chimera. In validation of germline transmission, donor PGC-derived progeny production efficiency was 94.68%, and the transgene production rate of heterozygous transgenic chickens was significantly increased in chickens that received 40 mg/kg busulfan (80.33-95.23%) compared with that of non-treated germline chimeras (51.18%). This system is expected to significantly improve the efficiency of generating transgenic chickens and other animal species by increasing the distribution of donor cells in adult testes.

Synthesis of 2-Chloro-3-amino indenone derivatives and their evaluation as inhibitors of DNA dealkylation repair.

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli AlkB, are involved in protecting DNA from alkylation damage. Inspired by the natural product indenone derivatives reported to inhibit this class of enzymes, and a set of 2-chloro-3-amino indenone derivatives was synthesized and screened for their inhibitory properties against AlkB. The synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones through addition-elimination method using alkyl/aryl amines under catalyst-free conditions. Using an in vitro reconstituted DNA repair assay, we have identified a 2-chloro-3-amino indenone compound 3o to be an inhibitor of AlkB. We have determined the binding affinity, mode of interaction, and kinetic parameters of inhibition of 3o and tested its ability to sensitize cells to methyl methanesulfonate that mainly produce DNA alkylation damage. This study established the potential of indenone-derived compounds as inhibitors of Fe(II)/2-oxoglutarate-dependent dioxygenase AlkB.