A Comparison of Body Size Evaluations of Obesity Surgery Patients and General Population Adults

RAND, COLLEEN S. W., JAQUELYN LISS RESNIK, AND ALEX M. C. MACGREGOR. A comparison of body size evaluations of obesity surgery patients and general population adults. Obes Res. Objective: To compare post-operative obesity surgery patients and general population adults in their assessments of a wide range of body sizes. Research Methods and Procedures: Obesity surgery patients (n = 274) and general population adults (n = 326) rated ideal and socially acceptable body sizes in separate arrays of babies, children, young adults, and middle-aged and older adults. Nine line figure drawings ranging from very thin to very obese were rated for each array. Results: Both groups selected the same ideal body size for all arrays except for babies. Both groups rejected obese and very thin body sizes as socially acceptable. However, the obesity surgery patients were more restrictive than general population adults in their ratings of socially acceptable body sizes. Current obesity status did not impact ratings for the patient or general population subjects. In the patient sample, time since surgery did not influence body size evaluations. Discussion: The study of body size ratings limited only to the “ideal” size may be misleading because it may mask subtle but meaningful differences between groups. The consistent difference in more restrictive ratings of obesity surgery patients compared to general population adults may be due to patients' greater psychological investment in endorsing the societal ideal body size. It may also be due to patients' status as peripheral group members of the normal weight community. The inability of some patients to maintain their post-operative weight loss may be particularly problematic for those who have defined “socially acceptable” body size most narrowly.     

The ecological integrity of the lower Olifants River,Limpopo province,South Africa: 2009–2015 – Part B: Tributaries of the Olifants River

Monitoring on the Lowveld reaches of the Olifants River, Limpopo River System, and its Steelpoort, Blyde, Klaserie and Selati tributaries was initiated in 2009. Analysis of the 2009–2015 data from four Olifants River sites showed deterioration in the river’s ecological condition between where it enters the Lowveld and where it enters the Kruger National Park, with a slight recovery within the Kruger National Park. Physico-chemical, aquatic macroinvertebrate and fish data collected in 2009–2015 at six sites on the Steelpoort, Blyde, Klaserie and Selati tributaries of the Olifants River corroborated the ecological condition of these tributaries. The Selati was the most polluted and was in a critically modified condition, whereas the Klaserie and Steelpoort were in fair condition and the Blyde was in good condition. The Selati appeared to have a significant negative impact on the water quality, macroinvertebrates and fish of the Olifants River within the Kruger National Park.     

Human health risks of metals and metalloids in muscle tissue of Synodontis zambezensis Peters, 1852 from Flag Boshielo Dam,South Africa

Muscle tissue from 63 Synodontis zambezensis collected bimonthly in 2013 at Flag Boshielo Dam were analysed for metals and metalloids in a desktop human health risk assessment. The Hazard Quotient, based on a weekly meal of 67 g of fish muscle, exceeded the maximum acceptable level of one for lead, cobalt, cadmium, mercury, arsenic and selenium. The concentrations of these elements were higher in 2013 than those recorded in 2009 and 2012 in other fish species from Flag Boshielo Dam and these may pose a long-term health risk if consumed regularly by impoverished rural communities reliant on fish as a source of protein.     

The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.Endogenous metabolism or environmental factors such as oxidizing and alkylating agents can produce a wide variety of lesions in DNA. The genomes of mammalian cells experience from 10,000 to as many as 200,000 modifications per day (37, 44). Most lesions are repaired by a complex network of proteins that are part of an elaborate, multistep base excision repair (BER) system that generates single-strand break (SSB) intermediates. Importantly, defects in BER can lead to malignancies and can be associated with age-associated disease, especially neurodegeneration (60).BER is initiated by specific DNA N-glycosylases that remove damaged bases, yielding apurinic/apyrimidinic (AP) sites. Subsequent incision by AP endonucleases results in SSBs, and excision results in a single base gap as a repair intermediate (33, 53). SSBs are expected to be frequent in the genome due to the abundance of base damage as well as intermediates of repair, recombination, replication, and other DNA transactions (15, 16). Because they are generally repaired efficiently by BER and SSB repair enzymes (16, 57), SSBs per se may not be a major source of genome instability. However, if lesions are clustered, the formation of two closely spaced SSBs on opposing strands (or a single SSB and a modified nucleotide or AP site) might pose a special risk in terms of the potential to generate mutations or the possibility of conversion to double-strand breaks (DSBs), which are potent genotoxic lesions. Clustered lesions can arise within cells by chance association of random DNA lesions in a small region or the induction of multiple events in a narrow region, as found for ionizing radiation and various chemicals, such as those used in cancer treatments (47, 58, 59). While efficient BER is important for genome integrity, the repair must be well coordinated to avoid the generation of closely opposed SSB intermediates at closely spaced lesions that could result in the secondary generation of DSBs, especially since cells have limited DSB repair capacity (<50 DSBs/cell in the case of Saccharomyces cerevisiae) (48). While the impact of clustered lesions on repair of DNA has been examined in vitro by use of purified enzymes or cell extracts (13, 14, 27, 39, 56), there has been little opportunity to address specifically the repair of clustered lesions, except for those arising from UV damage (49).Whether formed directly from sugar damage or as BER intermediates, SSBs formed during the repair of base damage often possess 5′-deoxyribose phosphate (5′-dRP) ends that are not suitable for rejoining by DNA ligases (9, 15). In humans, removal and repair of 5′-dRP are accomplished by different combinations of proteins (3, 15) that result in short-patch repair, involving replacement of a single nucleotide (nt), or long-patch repair, involving 2 to 10 nt. The budding yeast Saccharomyces cerevisiae lacks a DNA polymerase β that provides AP lyase activity required for short-patch repair in mammalian cells. Instead, removal and repair of a 5′-dRP rely on the long-patch pathway, involving the successive actions of DNA polymerase δ (Pol δ) for strand displacement, the Rad27/Fen1 endonuclease to remove 5′ flaps, and DNA ligase (Cdc9) to rejoin the resulting nicks (9). The sliding clamp protein PCNA, which interacts with all three players, has been proposed to play a central role in coordinating these processes (18, 19, 34). The coupling between the strand displacement reaction by Pol δ and the flap cutting reaction by Fen1 is highly efficient, with over 90% of the products released by Fen1 being mononucleotides (17).Although the coordination of Pol δ, PCNA, and Rad27/Fen1 provides efficient processing of individual lesions in DNA, closely opposed SSBs that arise during repair of base damage could manifest as DSBs, either directly or as a result of SSB processing. A DNA damaging agent that has been used frequently to characterize long- and short-patch BER is methyl methanesulfonate (MMS). Recently, we described the detection of closely opposed MMS-induced lesions in yeast (42). Since the closely opposed lesions might represent a special challenge to BER, we considered the possibility that they might specifically impact long-patch repair through Pol δ and/or coordination of events with Rad27/Fen1. Pol δ of S. cerevisiae is a heterotrimeric enzyme consisting of Pol3, Pol31, and Pol32 (23). The nonessential Pol32 subunit is involved in translesion DNA synthesis (TLS) (24, 30) and also break-induced replication (41). However, its role in other types of DNA repair remains unclear. Using our in vivo assay for specifically detecting closely spaced methylated DNA lesions (42) and SSBs, we examined the role of Pol32 as well as the cooperation between Pol δ, Rad27/Fen1, and PCNA in the repair of clustered DNA lesions induced by MMS in G₁ stationary-phase haploid yeast. We found that Pol32 plays an important role in ensuring that clustered lesions are efficiently repaired and do not transition to DSBs.     

Disruption of Supv3L1 damages the skin and causes sarcopenia, loss of fat, and death

Supv3L1 is a conserved and ubiquitously expressed helicase found in numerous tissues and cell types of many species. In human cells, SUPV3L1 was shown to suppress apoptotic death and sister chromatid exchange, and impair mitochondrial RNA metabolism and protein synthesis. In vitro experiments revealed binding of SUPV3L1 to BLM and WRN proteins, suggesting a role in genome maintenance processes. Disruption of the Supv3L1 gene in the mouse has been reported to be embryonic lethal at early developmental stages. We generated a conditional mouse in which the phenotypes associated with the removal of exon 14 can be tested in a variety of tissues. Disruption mediated by a Mx1 promoter-driven Cre displayed a postnatal growth delay, reduced lifespan, loss of adipose tissue and muscle mass, and severe skin abnormalities manifesting as ichthyosis, thickening of the epidermis, and atrophy of the dermis and subcutaneous tissue. Using a tamoxifen-activatable Esr1/Cre driver, Supv3L1 disruption resulted in growth retardation and aging phenotypes, including loss of adipose tissue and muscle mass, kyphosis, cachexia, and premature death. Many of the abnormalities seen in the Mx1-Cre mice, such as hyperkeratosis characterized by profound scaling of feet and tail, could also be detected in tamoxifen-inducible Cre mice. Conditional ablation of Supv3L1 in keratinocytes confirmed atrophic changes in the skin and ichthyosis-like changes. Together, these data indicate that Supv3L1 is important for the maintenance of the skin barrier. In addition, loss of Supv3L1 function leads to accelerated aging-like phenotypes.     

Broad Neutralization of Human Immunodeficiency Virus Type 1 (HIV-1) Elicited from Human Rhinoviruses That Display the HIV-1 gp41 ELDKWA Epitope

In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.The development of an AIDS vaccine is an ongoing and urgent challenge. One of the major hurdles is that the specific correlates of protection against human immunodeficiency virus (HIV) are still largely unknown. Nonetheless, most agree that the full complement of cellular and humoral components of the immune system will be needed to combat this virus. This is especially true given that the virus resides permanently in its host, infects the very cells needed to direct effective immune responses, and evades the immune system, either by changing in appearance or hiding in subcellular compartments.A broadly reactive neutralizing antibody response is likely to be critical as a first line of defense upon initial HIV exposure by aiding in the clearance of cell-free virions, targeting infected cells for destruction, and preventing viral spread through cell-to-cell transmission. The presence of inhibitory antibodies in highly exposed persistently seronegative individuals testifies to the importance of the humoral response (9, 37). Additionally, broadly neutralizing serum has been associated with healthier prognoses for infected individuals (27, 65) and may be vital for protecting offspring from their infected mothers (7, 79) and preventing superinfection by heterologous HIV strains (23, 84). Even if complete protection cannot be achieved by vaccine-derived antibodies, an early, well-poised and effective neutralizing antibody repertoire may be able to lower the set point of the viral load following the initial burst of viremia, an outcome that has been reported to translate into improved disease outcomes and reduced transmission of HIV (66, 74). Further benefits of neutralizing antibodies have been seen with passive immunization studies in macaques, in which administration of broadly neutralizing monoclonal antibodies (MAbs) has demonstrated that it is possible to provide protection from—and even sterilizing immunity against—HIV infection (5, 51, 66). There is also evidence that such antibodies may provide therapeutic benefits for chronically infected individuals, analogous to benefits realized with anti-HIV drug treatment regimens (87).Despite the promising potential of broadly neutralizing MAbs, designing immunogens that can elicit such cross-reactive neutralizing responses against HIV has been a surprisingly difficult task. Since the majority of the host''s B-cell response is directed against the envelope (Env) glycoproteins, gp120 and gp41, vaccine efforts have concentrated on these proteins and derivatives thereof in approaches ranging from the use of Env-based peptide cocktails to recombinant proteins and DNAs made with varied or consensus sequences and diverse, heterologous prime/protein boost regimens (reviewed in references 36, 58, and 70). These iterative studies have shown notable improvements in the potency and breadth of neutralizing responses induced. However, concerns exist regarding immunogens containing extraneous epitopes, as is the case with intact subunits of Env, and the nature of the immune responses they may elicit. A polyclonal burst of antibodies against a multitude of nonfunctional epitopes may include a predominance of antibodies that are (i) low affinity and/or nonfunctional (reviewed in reference 72); (ii) isolate specific (25); (iii) able to interfere with the neutralizing capabilities of otherwise-effective antibodies (via steric hindrance or by inducing various forms of B-cell pathology) (67); or (iv) directed against irrelevant epitopes instead of more conserved (and sometimes concealed) epitopes that might be able to elicit more potent and cross-reactive neutralizing responses (28, 71, 91).We have developed a system that can be used to present essentially any chosen epitope in a stable, well-exposed manner on the surface of the cold-causing human rhinovirus (HRV). HRV is itself a powerful immunogen and is able to elicit T-cell as well as serum and mucosal B-cell responses (reviewed by Couch [22]) and has minimal immunologic similarity to HIV (data not shown). Chimeric viruses displaying optimal epitopes should be able to serve as valuable components in an effective vaccine cocktail or as part of a heterologous prime/boost protocol. We have shown previously that HRV chimeric viruses displaying HIV-1 gp120 V3 loop sequences are able to elicit neutralizing responses against HIV-1 (75, 82, 83).In this study, we focused our attention on presenting part of the membrane-proximal external region (MPER) of the transmembrane glycoprotein gp41, a region of approximately 30 amino acids adjacent to the transmembrane domain (reviewed in references 59 and 97). The MPER plays an important role in the process of HIV fusion to the host cell membrane (60, 78). This region is also involved in binding to galactosylceramide, an important component of cell membranes, thus permitting CD4-independent transcytosis of the virus across epithelial cells at mucosal surfaces (1, 2). These functions likely explain this region''s sequence conservation and the efficacy of antibodies directed against the MPER (97), particularly given that an estimated 80% of HIV-1 infections are sexually transmitted at mucosal membranes. In fact, potent responses against the MPER are associated with stronger and broader neutralizing capabilities in infected individuals (68). A conserved, contiguous sequence of the MPER, the ELDKWA epitope (HIV-1 HxB2 gp41 residues 662 to 668), is recognized by the particularly broadly neutralizing human MAb 2F5 (11, 62, 85) and is highly resistant to escape mutation in the presence of 2F5 (49). 2F5 was also used in the MAb cocktails reported to confer passive, protective immunity in macaques (5, 51). In addition, infected individuals producing neutralizing antibodies directed against the ELDKWA epitope have been seen to exhibit better health (16, 29), including persistent seronegativity (8), and reduced transmission of HIV to offspring (89). While none of the vaccine-induced immune responses generated against this region has been effective thus far (19, 24, 26, 33, 35, 38, 40, 42, 44-48, 50, 53, 54, 56, 57, 61, 63, 69, 93, 96) (see Table S1 in the supplemental material), more appropriate presentations of MPER epitopes should produce valuable immunogens that can contribute to a successful vaccine.In this study, we have grafted the ELDKWA epitope onto a surface loop of HRV connected via linkers of variable lengths and sequences and selected for viruses well recognized and neutralized by MAb 2F5. In so doing, we have been able to create immunogens capable of eliciting antibodies whose activities mimic some of those of 2F5. The combinatorial libraries produced were designed to encode a large set of possible sequences and, hence, structures from which we could search for valuable conformations. This work illustrates that HRV chimeras have the potential to present selected HIV epitopes in a focused and immunogenic manner.     

Identification and SAR of squarate inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. These squarate based inhibitors were identified via a high-throughput screen. An MK2 co-structure with the starting ligand was obtained and a structure based approach was followed to optimize potency and selectivity.     

Mutations in the Chromosomal Passenger Complex and the Condensin Complex Differentially Affect Synaptonemal Complex Disassembly and Metaphase I Configuration in Drosophila Female Meiosis

Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. We characterized female-sterile alleles of the condensin complex component dcap-g and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, we demonstrate a role for dcap-g in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I-arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. We used the QA26 allele of passenger component incenp to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenp^QA26 mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient toward only one pole in prometaphase and metaphase I. We show that incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions.     

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.