Cultivating a Meaningful Application of IMFs through Backward Laboratory Course Design.

This paper describes the development of a first- and second-year inquiry-based laboratory course focused on the development of a meaningful application of intermolecular forces (IMFs). Instead of broad expository coverage of topics, we used backward design: the techniques and concepts for the course were structured around what students are expected to be able to do at the end-individually isolate caffeine from a consumer product as a culminating lab practical, using IMFs to justify solvent choices and determining procedural details. We have found that instructors can select a challenging multilevel experiment that incorporates the application of IMFs in multiple ways and backward design the course so that students are able to complete this experiment individually and autonomously at the end of the semester. By incorporating evidence-based pedagogies to foster meaningful learning and repetition of techniques and IMF concepts in different contexts, we promoted opportunities to learn from mistakes and prioritized student decision making. This approach involved faculty collaboration and spanned several semesters of iteration. In our experience, a cumulative lab practical motivates students to learn the techniques and take responsibility for learning. We propose that the backward design process with a central theme, such as the application of IMFs in our case, is especially well suited to planning a chemistry laboratory course. However, even with an entire laboratory course centered around applications of this critical concept, we discovered there were still gaps in students' abilities to apply IMFs.

Non-target effect of bispyribac sodium on soil microbial community in paddy soil.

Bispyribac sodium is frequently used herbicide in the rice field. Though, it has been targeted to kill rice weeds, but its non-target effect on soil microbes in paddy soil was largely unknown. Therefore, in the present study, an attempt was made to assess the non-target effect of bispyribac sodium on alteration of functional variation of soil microbial community and their correlation with microbial biomass carbon (MBC) and soil enzymes. A microcosm experiment set up was made comprising three treatments viz., control (CON) (without application of bispyribac sodium), recommended dose of bispyribac sodium (35 g ha-1) (BS), and double the dose of BS (70 g ha-1) (DBS). Results indicated that the MBC and soil enzyme activities (dehydrogenase, alkaline phosphatase and urease) in BS and DBS-treated soil were significantly (p < 0.05) declined from 1st to 30th day after application as compared to CON. Counts of heterotrophic bacteria, actinomycetes and fungal population were also decreased in BS and DBS-treated soil. The average well color development (AWCD) values derived from Biolog®ecoplates followed the order of DBS ˂ BS ˂ CON. Shannon index value was high (p ≤ 0.05) in CON compared to soil-treated with BS and DBS. Principal component analysis (PCA) showed a clear distinction of the cluster of treatments between CON, BS and DBS. Biplot analysis and heatmap suggested that carboxylic compounds and amino acids showed positive response towards BS-treated soil, whereas phenolic compounds had positive correlation with DBS-treated soil. PCA analysis indicated that oligotrophs was rich in BS-treated paddy soil, whereas copiotrophs and asymbiotic nitrogen fixers were richer in DBS treatment. Overall, the present study revealed that application of recommended dose of BS and its double dose alter the soil microbial population, enzyme activities and functional microbial diversity in paddy soil.

Mechanism of frataxin "bypass" in human iron-sulfur cluster biosynthesis with implications for Friedreich's ataxia.

In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.

Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7.

Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5-10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors.

Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.

Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR.

Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic Pathways.

Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.

A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells.

Our prior study utilized both in vitro and in vivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells.

Synergistic anti-myeloma activity of the proteasome inhibitor marizomib and the IMiD immunomodulatory drug pomalidomide.

The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action and effects on proteasomal activities, and that it can overcome bortezomib resistance. Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that combining low concentrations of marizomib with pomalidomide induces synergistic anti-MM activity. Marizomib plus pomalidomide-induced apoptosis is associated with: (i) activation of caspase-8, caspase-9, caspase-3 and PARP cleavage, (ii) downregulation of cereblon (CRBN), IRF4, MYC and MCL1, and (iii) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. CRBN-siRNA attenuates marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and tumour-associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow microenvironment. In human MM xenograft model studies, the combination of marizomib and pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical studies provide the rationale for on-going clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM.

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe-S Assembly Complex.

Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.

Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT/ß-CATENIN signaling for Sertoli cell mediated regulation of spermatogenesis.

Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4-6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/ß-CATENIN activity. Elevated ß-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/ß-CATENIN modulation.

Role of apoptosis-inducing factor (Aif) in the T cell lineage.

Multiple checkpoints regulating finely balanced death-versus-survival decisions characterize both thymic development and peripheral homeostasis of T lymphocytes. While exploring the mechanisms of T cell death involved at various stages during the life of a T cell, we have observed and reported a variety of non-redundant roles for apoptosis inducing factor (Aif), a mitochondrial flavoprotein. Aif is ubiquitously expressed in all cell lineages and functions as an NADH oxidase in its mitochondrial location. It is released following the mitochondrial death signals, whereupon it translocates to the nucleus, binds to DNA and causes large-scale DNA fragmentation. During T cell development, Aif is important for developing thymocytes to navigate the double negative (DN)3 to DN4 transition (beta-selection), via its oxidoreductase property which protects the rapidly proliferating cells from death due to reactive oxygen species (ROS). In peripheral mature T cells, Aif deficiency leads to an increased susceptibility of T cell blasts to activation induced cell death (AICD), possibly mediated by its antioxidant function, and decreased sensitivity to neglect-induced death (NID). Thus, Aif seems to have pro-apoptotic and anti-apoptotic roles in the same lineage in different contexts and at different stages. Surprisingly, in the closely related B lymphocyte lineage, Aif deficiency does not result in any abnormality. These findings generate the possibility of specific T cell dysfunction in human disease caused by Aif deficiency, as well as in mitochondriopathies due to other causes. Also, these data raise questions regarding the basis of lineage-specific consequences of the dysfunction/deficiency of apparently ubiquitous molecules.

A role for apoptosis-inducing factor in T cell development.

Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein that regulates cell metabolism and survival in many tissues. We report that aif-hypomorphic harlequin (Hq) mice show thymic hypocellularity and a cell-autonomous thymocyte developmental block associated with apoptosis at the ß-selection stage, independent of T cell receptor ß recombination. No abnormalities are observed in the B cell lineage. Transgenes encoding wild-type or DNA-binding-deficient mutant Aif rectify the thymic defect, but a transgene encoding oxidoreductase activity-deficient mutant Aif does not. The Hq thymic block is reversed in vivo by antioxidant treatment, and Hq T but not B lineage cells show enhanced oxidative stress. Thus, Aif, a ubiquitous protein, serves a lineage-specific nonredundant antiapoptotic role in the T cell lineage by regulating reactive oxygen species during thymic ß-selection.