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Human mortalin (HSPA9) was originally identified by its close homology to murine mortalins, which play important roles in cellular senescence. The two murine genes, mot-1 and mot-2, differ in only two amino acid residues, but have opposite functions in cellular immortalization. HSPA9 was recently localized to chromosome 5, band q31, a region that is frequently deleted in myeloid leukemias and myelodysplasia (MDS), making it a candidate tumor suppressor gene, which is consistent with the biological function of its murine homologue. To evaluate mortalin in this capacity, its expression in normal and leukemic cell lines was investigated, and its genomic structure was determined in order to facilitate mutation detection. RT-PCR and Northern blot analysis revealed a broad distribution in normal tissues and in leukemia cell lines, producing a single 2.8 kb transcript. Genomic characterization showed that the gene spans 18 kb, and consisted of 17 exons with boundaries that were almost identical to its murine counterpart. Using intron-based primers to flank each exon, sequence of the complete protein-coding regions was obtained for three AML cell lines, including two lines with chromosome 5 loss (KG-1 and HL-60) and one without (AML-193) compared to normal DNA. No mutations were identified although one conservative nucleotide sequence variant was observed in exon 16. We have shown that mortalin is highly conserved in genomic structure as well as sequence, and the designed primers will be suitable for future studies to detect mutations in clinical samples. [ABSTRACT FROM AUTHOR]

AML with chromosomal alterations involving 3q26 overexpresses the transcription factor (TF) EVI1, associated with therapy refractoriness and inferior overall survival in AML. Consistent with a CRISPR screen highlighting BRD4 dependency, treatment with BET inhibitor (BETi) repressed EVI1, LEF1, c-Myc, c-Myb, CDK4/6, and MCL1, and induced apoptosis of AML cells with 3q26 lesions. Tegavivint (TV, BC-2059), known to disrupt the binding of nuclear β-catenin and TCF7L2/LEF1 with TBL1, also inhibited co-localization of EVI1 with TBL1 and dose-dependently induced apoptosis in AML cell lines and patient-derived (PD) AML cells with 3q26.2 lesions. TV treatment repressed EVI1, attenuated enhancer activity at ERG, TCF7L2, GATA2 and MECOM loci, abolished interactions between MYC enhancers, repressing AML stemness while upregulating mRNA gene-sets of interferon/inflammatory response, TGF-β signaling and apoptosis-regulation. Co-treatment with TV and BETi or venetoclax induced synergistic in vitro lethality and reduced AML burden, improving survival of NSG mice harboring xenografts of AML with 3q26.2 lesions., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

We earlier reported substantial progress in designing gp120 antagonists. Notably, we discovered that NBD-14189 is not only the most active gp120 antagonist but also shows antiviral activity against HIV-1 Reverse Transcriptase (RT). We also confirmed its binding to HIV-1 RT by X-ray crystallography. The dual inhibition is highly significant because, intriguingly, this compound bridges the dNTP and NNRTI-binding sites and inhibits the polymerase activity of isolated RT in the enzymatic assay. This novel finding is expected to lead to new avenues in designing a novel class of HIV-1 dual inhibitors. Therefore, we needed to advance this inhibitor to preclinical assessment. To this end, we report the pharmacokinetics (PK) study of NBD-14189 in rats and dogs. Subsequently, we assessed the toxicity and therapeutic efficacy in vivo in the SCID-hu Thy/Liv mouse model. The PK data indicated a favorable half-life (t 1/2 ) and excellent oral bioavailability (%F = 61%). NBD-14189 did not show any measurable toxicity in the mice, and treatment reduced HIV replication at 300 mg/kg per day in the absence of clear evidence of protection from HIV-mediated human thymocyte depletion. The data indicated the potential of this inhibitor as an anti-HIV-1 agent and needs to be assessed in a non-human primate (NHP) model.

Use of nicotine-specific monoclonal antibodies (mAbs) to sequester and reduce nicotine distribution to brain has been proposed as a therapeutic approach to treat nicotine addiction (the basis of tobacco use disorder). A series of monoclonal antibodies with high affinity for nicotine (nic•mAbs) was isolated from B-cells of vaccinated smokers. Genes encoding 32 unique nicotine binding antibodies were cloned, and the mAbs expressed and tested by surface plasmon resonance to determine their affinity for S-(-)-nicotine. The highest affinity nic•mAbs had binding affinity constants (KD) between 5 and 67 nM. The 4 highest affinity nic•mAbs were selected to undergo additional secondary screening for antigen-specificity, protein properties (including aggregation and stability), and functional in vivo studies to evaluate their capacity for reducing nicotine distribution to brain in rats. The 2 most potent nic•mAbs in single-dose nicotine pharmacokinetic experiments were further tested in a dose-response in vivo study. The most potent lead, ATI-1013, was selected as the lead candidate based on the results of these studies. Pretreatment with 40 and 80 mg/kg ATI-1013 reduced brain nicotine levels by 56 and 95%, respectively, in a repeated nicotine dosing experiment simulating very heavy smoking. Nicotine self-administration was also significantly reduced in rats treated with ATI-1013. A pilot rat 30-day repeat-dose toxicology study (4x200mg/kg ATI-1013) in the presence of nicotine indicated no drug-related safety concerns. These data provide evidence that ATI-1013 could be a potential therapy for the treatment of nicotine addiction., Competing Interests: At the time the research was conducted: i) TT, ZB, and MK are/were employees and shareholders of Antidote Therapeutics, Inc. (ATI), ii) S. Fuller was a consultant to ATI and shareholder, iii) NB and S. Fallot were employees and shareholders of Blink Biomedical, SAS, iv) DS was an employee of BioFactura, Inc. and ATI shareholder, v) SH was an employee of Noble Life Sciences, Inc. MK, TT, NB, S. Fallot, ZB, S. Fuller, MLS, and PP are coinventors on the following patent application: “Novel Nicotine-binding Antibodies” US201816639050 20180814. MR, NB, S. Fallot, MLS, AS, PP, S. Fuller, SH, DS, and BZ and their respective institutions received research funding from Antidote for salaries, experiments, and manufacturing as subawardees1,5,6 or subcontractors3,4 to Antidote under R01 DA038877 from the National Institute on Drug Abuse (NIDA) at the National Institutes of Health.

Transformation of post-myeloproliferative neoplasms into secondary (s) AML exhibit poor clinical outcome. In addition to increased JAK-STAT and PI3K-AKT signaling, post-MPN sAML blast progenitor cells (BPCs) demonstrate increased nuclear β-catenin levels and TCF7L2 (TCF4) transcriptional activity. Knockdown of β-catenin or treatment with BC2059 that disrupts binding of β-catenin to TBL1X (TBL1) depleted nuclear β-catenin levels. This induced apoptosis of not only JAKi-sensitive but also JAKi-persister/resistant post-MPN sAML BPCs, associated with attenuation of TCF4 transcriptional targets MYC, BCL-2, and Survivin. Co-targeting of β-catenin and JAK1/2 inhibitor ruxolitinib (rux) synergistically induced lethality in post-MPN sAML BPCs and improved survival of mice engrafted with human sAML BPCs. Notably, co-treatment with BET protein degrader ARV-771 and BC2059 also synergistically induced apoptosis and improved survival of mice engrafted with JAKi-sensitive or JAKi-persister/resistant post-MPN sAML cells. These preclinical findings highlight potentially promising anti-post-MPN sAML activity of the combination of β-catenin and BETP antagonists against post-MPN sAML BPCs.

Purpose: We compared the rate and severity of fatigue in patients who completed stereotactic body radiation therapy (SBRT) to the liver daily (QD) compared with every other day (QOD)., Methods and Materials: From 2010 to 2017, 91 patients with Child Pugh (CP) A (n = 57) or CP-B (n = 34) cirrhosis who completed 100 SBRT sessions to 110 hepatocellular carcinoma (HCC) lesions were analyzed in this study. Confounding variables with fatigue such as CP-C cirrhosis, Eastern Cooperative Oncology Group score >2, or a history of ascites or encephalopathy were excluded. Fatigue was assessed against several treatment- and patient-related variables with univariate and propensity score-matched multivariate analysis. The median follow-up time was 18 months., Results: Patients with HCC and Barcelona-Clinic Liver Cancer stages 0 (n = 10), A (n = 32), and B (n = 58), and a median age of 62 years were analyzed. The median tumor diameter was 3 cm (1.1-11 cm). The Eastern Cooperative Oncology Group performance status score was 0 (n = 44), 1 (n = 43), or 2 (n = 13). The median dose was 45 Gy in 5 fractions, and 65 treatments were QD and 45 QOD. Grades 1 and 2 fatigue developed in 49% and 14% of treatments, respectively. Among the patients who were treated daily, 78% developed Grade 1 or 2 fatigue compared with 44% who were treated QOD (odds ratio: 4.52; P = .001). Grade 2 fatigue occurred in 22% of patients compared with 7.3% for QD and QOD treatment, respectively (odds ratio: 3.83; P = .048). There was no difference in fatigue rate for time of treatment (morning or afternoon), dose, treated volume, CP score, Barcelona-Clinic Liver Cancer stage, or performance status, which were not associated with any level of fatigue. There was no difference in local control between QD and QOD treatments., Conclusions: Compared with traditional daily treatment fractions, SBRT that is delivered QOD to cirrhotic patients with HCC may reduce the risk of fatigue., (Copyright © 2018 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

Background: The bacterial nicotine-degrading enzyme NicA2 isolated from P. putida was studied to assess its potential use in the treatment of tobacco dependence., Results: Rats were pretreated with varying i.v. doses of NicA2, followed by i.v. administration of nicotine at 0.03 mg/kg. NicA2 had a rapid onset of action reducing blood and brain nicotine concentrations in a dose-related manner, with a rapid onset of action. A 5 mg/kg NicA2 dose reduced the nicotine concentration in blood by > 90% at 1 min after the nicotine dose, compared to controls. Brain nicotine concentrations were reduced by 55% at 1 min and 92% at 5 min post nicotine dose. To evaluate enzyme effects at a nicotine dosing rate equivalent to heavy smoking, rats pretreated with NicA2 at 10 mg/kg were administered 5 doses of nicotine 0.03 mg/kg i.v. over 40 min. Nicotine levels in blood were below the assay detection limit 3 min after either the first or fifth nicotine dose, and nicotine levels in brain were reduced by 82 and 84%, respectively, compared to controls. A 20 mg/kg NicA2 dose attenuated nicotine discrimination and produced extinction of nicotine self-administration (NSA) in most rats, or a compensatory increase in other rats, when administered prior to each daily NSA session. In rats showing compensation, increasing the NicA2 dose to 70 mg/kg resulted in extinction of NSA. An enzyme construct with a longer duration of action, via fusion with an albumin-binding domain, similarly reduced NSA in a 23 h nicotine access model at a dose of 70 mg/kg., Conclusions: These data extend knowledge of NicA2's effects on nicotine distribution to brain and its ability to attenuate addiction-relevant behaviors in rats and support its further investigation as a treatment for tobacco use disorder.

Currently available treatment options are unlikely to be curative for the majority of multiple myeloma patients, emphasizing a continuing role for the introduction of investigational agents that can overcome drug resistance. The canonical Wnt/β-catenin signaling pathway, essential for self-renewal, growth, and survival, has been found to be dysregulated in multiple myeloma, particularly in advanced stages of disease. This provides the rationale for evaluating the novel β-catenin inhibitor BC2059 as monotherapy and in combination with proteasome inhibitors in vitro and in vivo Here, we show nuclear localization of β-catenin in human myeloma cell lines (HMCL), consistent with activation of the canonical Wnt pathway. BC2059 attenuates β-catenin levels, in both the cytoplasm and the nucleus, reducing the transcriptional activity of the TCF4/LEF complex and the expression of its target gene axin 2. Treatment of HMCL with BC2059 inhibits proliferation and induces apoptosis in a dose-dependent manner. This is also observed in HMCL-stromal cell cocultures, mitigating the protective effect afforded by the stroma. Similarly, BC2059 induces apoptosis in primary multiple myeloma samples in vitro , causing minimal apoptosis on healthy peripheral blood mononuclear cells. Furthermore, it synergizes with the proteasome inhibitor bortezomib both in HMCL and primary multiple myeloma samples. Finally, in xenograft models of human myelomatosis, BC2059 delays tumor growth and prolongs survival with minor on-target side effects. Collectively, these results demonstrate the efficacy of targeting the Wnt/β-catenin pathway with BC2059 both in vitro and in vivo , at clinically achievable doses. These findings support further clinical evaluation of BC2059 for the treatment of multiple myeloma. Mol Cancer Ther; 16(9); 1765-78. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Objective: To evaluate the efficacy of a brief cognitive-behavioral therapy (CBT) that is being developed for management of cognitive dysfunction following chemotherapy among breast cancer survivors. Memory and Attention Adaptation Training (MAAT) is a brief CBT designed to improve the quality of life and function among cancer survivors with post-chemotherapy cognitive complaints., Methods: An initial, two-group (MAAT versus waitlist, no treatment control), randomized clinical trial (RCT) was conducted. Forty stage I and II female breast cancer survivors (mean age = 50; SD = 6.4) were randomized to conditions and assessed at baseline, post-treatment (8 weeks) and 2-month follow-up assessment points on measures of: (1) self-reported daily cognitive failures; (2) quality of life; and (3) neuropsychological performance. Participants were also assessed for satisfaction with MAAT., Results: With education and IQ as covariates, MAAT participants made significant improvements relative to controls on the spiritual well-being subscale of the quality of life measure and on verbal memory, but statistical significance was not achieved on self-report of daily cognitive complaints. However, moderate-to-large effect sizes were observed on these outcomes. Participants gave MAAT high satisfaction ratings., Conclusions: Although this initial RCT is a small study, MAAT participants appear to improve on one measure of quality of life and verbal memory performance relative to no treatment controls and rate MAAT with high satisfaction. These data are encouraging and support the continued development and evaluation of MAAT efficacy., (Copyright © 2010 John Wiley & Sons, Ltd.)

Following the emergence of Salmonella Enteritidis as a widespread contaminant of eggs and the role of eggs in the transmission of human salmonellosis, control measures were introduced to curb the spread of infection. Two approaches to Salmonella control are currently used by egg producers in Ireland, because Northern Ireland producers, like those in the rest of the United Kingdom, widely adopted a vaccination regime, whereas the Republic of Ireland does not permit vaccination but introduced controls based on routine monitoring for specific Salmonella serovars and subsequent culling of infected flocks. To compare the efficacy of these two approaches and determine the prevalence of salmonellae in eggs produced for retail sale in the island of Ireland, a major survey of approximately 30,000 grade A eggs was undertaken. Egg shells and contents were analyzed separately for salmonellae by procedures based on International Organization for Standardization methodology. The survey yielded only two positive samples, with Salmonella Infantis and Salmonella Montevideo isolated from shells; no egg contents yielded salmonellae. There was no statistically significant difference in the prevalence of salmonellae between eggs produced in Northern Ireland and those from the Republic of Ireland; hence, both regimes appeared to be equally effective in controlling salmonellae. The prevalence was also significantly lower than that found in a recent major United Kingdom survey. Hence, shell eggs produced in the island of Ireland are unlikely to be a source of human salmonellosis.

Interstitial deletion or loss of chromosome 5, del(5q) or -5, is a frequent finding in myeloid leukemias and myelodysplasias, suggesting the presence of a tumor suppressor gene within the deleted region. In our search for this gene, we identified a candidate, 5qNCA (LOC51780), which lies within a consistently-deleted segment of 5q31. 5qNCA expresses a 7.2-kb transcript with a 5286-bp open reading frame which is present at high levels in heart, skeletal muscle, kidney, placenta, and liver as well as CD34+ cells and AML cell lines. 5qNCA encodes a 191-kD nuclear protein which contains a highly-conserved C-terminus containing a zinc finger with the unique spacing Cys-X2-Cys-X7-His-X2-Cys-X2-Cys-X4-Cys-X2-Cys and a jmjC domain, which is often found in proteins that regulate chromatin remodeling. Expression of 5qNCA in a del(5q) cell line results in suppression of clonogenic growth. Preliminary sequence results in AML and MDS samples and cell lines has revealed a possible mutation in the KG-1 cell line resulting in a THR to ALA substitution that has not been found in over 100 normal alleles to date. We propose 5qNCA is a good candidate for the del(5q) tumor suppressor gene based on its predicted function and growth suppressive activities, and suggest that further mutational and functional study of this interesting gene is warranted.

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

Interstitial deletion or loss of chromosome 5 is frequent in malignant myeloid disorders, including myelodysplasia (MDS) and acute myeloid leukemia (AML), suggesting the presence of a tumor suppressor gene. Loss of heterozygosity (LOH) analysis was used to define a minimal deletion interval for this gene. Polymorphic markers on 5q31 were identified using a high-resolution physical and radiation hybrid breakpoint map and applied to a patient with AML with a subcytogenetic deletion of 5q. By comparing the DNA from leukemic cells to buccal mucosa cells, LOH was detected with markers D5S476 and D5S1372 with retention of flanking markers D5S500 to D5S594. The D5S500-D5S594 interval, which covers approximately 700 kb, thus represents a minimal localization for the tumor suppressor gene. Further refinement of the physical map enabled the specification of 9 transcription units within the encompassing radiation hybrid bins and 7 in flanking bins. The 9 candidates include genes CDC25, HSPA9, EGR1, CTNNA1, and 5 unknown ESTs. Reverse-transcription polymerase chain reaction confirms that all of them are expressed in normal human bone marrow CD34(+) cells and in AML cell lines and thus represent likely candidates for the MDS-AML tumor suppressor gene at 5q31.

We have constructed a high-resolution map of a 6-Mb interval of human chromosome 5, band q31, incorporating 175 sequence tagged sites, of which 33 are genetic polymorphisms and 122 are nonredundant expressed sequences. The map was assembled initially as a YAC contig, incorporating data from radiation hybrid maps. To improve resolution and to identify errors in the databases, a radiation hybrid breakpoint map was developed for the interval, which included hybrids from both Stanford G3 and GeneBridge 4 panels. This novel approach facilitated the integration of one RH panel with another and enabled the identification and localization of new, previously unmapped ESTs from the radiation hybrid databases. ESTs were assembled into overlapping transcription units and ordered with respect to polymorphic markers in the region, resulting in a comprehensive map that incorporates markers from multiple different types of maps. This map of 5q31 will facilitate gene discovery efforts for several disorders, including limb-girdle muscular dystrophy type 1A and the genes deleted in acute myeloid leukemias and myelodysplasia. The study demonstrates the utility of a radiation hybrid breakpoint panel for correction of map errors and for the efficient identification of new transcript units in a large genomic interval., (Copyright 1999 Academic Press.)

Limb-girdle muscular dystrophy type 1A (LGMD1A) is an autosomal dominant disease characterized by progressive weakness of the hip and shoulder girdle. The gene for LGMD1A had been localized to a 7-cM interval at 5q31 in a single large family (Family 39). To refine the localization of LGMD1A further and to aid in its identification, a high-resolution physical map of the locus was used to identify and provisionally localize 25 polymorphic markers. A subset of these markers was then ordered genetically, using a CEPH meiotic breakpoint panel, resulting in an integrated physical-genetic map of the locus. Relevant markers were genotyped on the members of Family 39 who contained informative recombination events, resulting in a further narrowing of LGMD1A to an interval bounded by D5S479 and D5S594, estimated to be 2 Mb in size. Integration of the genetic and physical map permits the identification of several transcription units from within the narrowed LGMD1A interval, including one that is muscle specific, representing candidate genes for this familial dystrophy., (Copyright 1998 Academic Press.)

The limb-girdle muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Recent linkage analyses and positional cloning studies have identified numerous loci responsible for the recessive and dominant forms, underscoring the inherent heterogeneity. In this report, we investigate four large autosomal dominant limb-girdle pedigrees and exclude these pedigrees from linkage to these loci. In addition, there is no evidence for linkage to any of the seven recessive LGMD loci.