A higher order PUF complex is central to regulation of C. elegans germline stem cells.

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including gld-1. Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses gld-1 via a pair of adjacent FBF-2 binding elements (FBEs) in its 3ÚTR. One LST-1 molecule links two FBF-2 molecules via motifs in the LST-1 intrinsically-disordered region; the gld-1 FBE pair includes a well-established 'canonical' FBE and a newly-identified noncanonical FBE. Remarkably, this FBE pair drives both full RNA repression in GSCs and full RNA activation upon differentiation. Discovery of the LST-1-FBF-2 ternary complex, the gld-1 adjacent FBEs, and their in vivo significance predicts an expanded regulatory repertoire of different assemblies of PUF-partner complexes in nematode germline stem cells. It also suggests analogous PUF controls may await discovery in other biological contexts and organisms.

LST-1 is a bifunctional regulator that feeds back on Notch-dependent transcription to regulate C. elegans germline stem cells.

Notch signaling regulates stem cells across animal phylogeny. C. elegans Notch signaling activates transcription of two genes, lst-1 and sygl-1, that encode potent regulators of germline stem cells. The LST-1 protein regulates stem cells in two distinct ways: It promotes self-renewal posttranscriptionally and also restricts self-renewal by a poorly understood mechanism. Its self-renewal promoting activity resides in its N-terminal region, while its self-renewal restricting activity resides in its C-terminal region and requires the Zn finger. Here, we report that LST-1 limits self-renewal by down-regulating Notch-dependent transcription. We detect LST-1 in the nucleus, in addition to its previously known cytoplasmic localization. LST-1 lowers nascent transcript levels at both lst-1 and sygl-1 loci but not at let-858, a Notch-independent locus. LST-1 also lowers levels of two key components of the Notch activation complex, the LAG-1 DNA binding protein and Notch intracellular domain (NICD). Genetically, an LST-1 Zn finger mutant increases Notch signaling strength in both gain- and loss-of-function GLP-1/Notch receptor mutants. Biochemically, LST-1 co-immunoprecipitates with LAG-1 from nematode extracts, suggesting a direct effect. LST-1 is thus a bifunctional regulator that coordinates posttranscriptional and transcriptional mechanisms in a single protein. This LST-1 bifunctionality relies on its bipartite protein architecture and is bolstered by generation of two LST-1 isoforms, one specialized for Notch downregulation. A conserved theme from worms to human is the coupling of PUF-mediated RNA repression together with Notch feedback in the same protein.

The in vivo functional significance of PUF hub partnerships in C. elegans germline stem cells.

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of Caenorhabditis elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we previously proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1-PUF and SYGL-1-PUF partnerships and their molecular activities in their natural context - nematode stem cells. We confirm LST-1-PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the in vivo functional significance of the LST-1-PUF partnership. Tethered LST-1 requires this partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs in vivo. Comparison of LST-1-PUF and Nanos-Pumilio reveals fundamental molecular differences, making LST-1-PUF a distinct paradigm for PUF partnerships.

Functional significance of PUF partnerships in C. elegans germline stem cells.

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of C. elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1-PUF and SYGL-1-PUF partnerships and their molecular activities in their natural context - nematode stem cells. We confirm LST-1-PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(A m B m ) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(A m B m ) is used to explore the functional significance of the LST-1-PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs. Comparison of PUF-LST-1 and Pumilio-Nanos reveals fundamental molecular differences, making PUF-LST-1 a distinct paradigm for PUF partnerships. Summary statement: Partnerships between PUF RNA-binding proteins and intrinsically disordered proteins are essential for stem cell maintenance and RNA repression.

Development of a novel screening tool for predicting Cochlear implant candidacy.

OBJECTIVES: Cochlear implantation (CI) is a well-established treatment for sensorineural hearing loss. Due in part to a lack of referral guidelines, CI technology remains underutilized, and many patients who could benefit from CI may not be referred for evaluation. This study aimed to develop a model for predicting CI candidacy using routine audiometric measures, with the goal of providing guidance to clinicians regarding when to refer a patient for CI evaluation. METHODS: Unaided three-frequency pure tone average (PTA), unaided speech discrimination score (SDS), and best-aided sentence recognition testing with AZBio sentence lists were collected from 252 subjects undergoing CIE. Candidacy was defined by meeting traditional (AZBio score ≤ 60%), or Medicare criteria (≤40%). A logistic regression model was developed to predict candidacy. Confusion matrices were plotted to determine the sensitivity and specificity at various probability thresholds. RESULTS: Logistic regression models were capable of predicting probability of candidacy for traditional criteria (P < .001) and Medicare criteria (P < .001). PTA and SDS were significant predictors (P < .001). Using a probability cutoff of .5, the models yielded a sensitivity rate of 91% and 78% for traditional and Medicare criteria, respectively. CONCLUSION: Probability of CI candidacy may be determined using a novel screening tool for referral. This tool supports individualized counseling, serves as a proof of concept for candidacy prediction, and could be modified based on an institution's philosophy regarding an acceptable false positive rate of referral. LEVEL OF EVIDENCE: 4.

In vivo delivery of synthetic DNA-encoded antibodies induces broad HIV-1-neutralizing activity.

Interventions to prevent HIV-1 infection and alternative tools in HIV cure therapy remain pressing goals. Recently, numerous broadly neutralizing HIV-1 monoclonal antibodies (bNAbs) have been developed that possess the characteristics necessary for potential prophylactic or therapeutic approaches. However, formulation complexities, especially for multiantibody deliveries, long infusion times, and production issues could limit the use of these bNAbs when deployed, globally affecting their potential application. Here, we describe an approach utilizing synthetic DNA-encoded monoclonal antibodies (dmAbs) for direct in vivo production of prespecified neutralizing activity. We designed 16 different bNAbs as dmAb cassettes and studied their activity in small and large animals. Sera from animals administered dmAbs neutralized multiple HIV-1 isolates with activity similar to that of their parental recombinant mAbs. Delivery of multiple dmAbs to a single animal led to increased neutralization breadth. Two dmAbs, PGDM1400 and PGT121, were advanced into nonhuman primates for study. High peak-circulating levels (between 6 and 34 µg/ml) of these dmAbs were measured, and the sera of all animals displayed broad neutralizing activity. The dmAb approach provides an important local delivery platform for the in vivo generation of HIV-1 bNAbs and for other infectious disease antibodies.

Worksite wellness program implementation: a model of translational effectiveness.

Occupational health promotion programs with documented efficacy have not penetrated worksites. Establishing an implementation model would allow focusing on mediating aspects to enhance installation and use of evidence-based occupational wellness interventions. The purpose of the study was to implement an established wellness program in fire departments and define predictors of program exposure/dose to outcomes to define a cross-sectional model of translational effectiveness. The study is a prospective observational study among 12 NW fire departments. Data were collected before and following installation, and findings were used to conduct mediation analysis and develop a translational effectiveness model. Worker age was examined for its impact. Leadership, scheduling/competing demands, and tailoring were confirmed as model components, while organizational climate was not a factor. The established model fit data well (χ (2)(9) = 25.57, CFI = 0.99, RMSEA = 0.05, SRMR = 0.03). Older firefighters, nearing retirement, appeared to have influences that both enhanced and hindered participation. Findings can inform implementation of worksite wellness in fire departments, and the prioritized influences and translational model can be validated and manipulated in these and other settings to more efficiently move health promotion science to service.

Persistence of unrepaired DNA double strand breaks caused by inhibition of ATM does not lead to radio-sensitisation in the absence of NF-κB activation.

The stress-inducible transcription complex NF-κB induces the transcription of genes that regulate proliferation and apoptosis. Constitutively activated NF-κB is common in breast cancers, and contributes to malignant progression and therapeutic resistance. Ataxia telangiectasia mutated (ATM) is a key regulator of the cellular response to DNA double strand breaks (DSBs), and recent reports have demonstrated that ATM is required for the activation of NF-κB following DNA damage. We investigated the role of ATM in the NF-κB signalling cascade induced by ionising radiation (IR) in breast cancer cell lines using KU55933, a novel and specific inhibitor of ATM. KU55933 suppressed IR-induced IκBα degradation, p50/p65 nuclear translocation and binding to kB consensus sequences. KU55933 also suppressed transcription of an NF-κB dependent reporter gene and inhibited IR-induced DSB repair as assessed by the neutral Comet assay. KU55933 sensitised cells to IR, with a concurrent increase in caspase 3 activity. Importantly, KU55933 sensitised IKKß(+/+) and p65(+/+), but not IKKß(-/-) or p65(-/-), mouse embryonic fibroblasts to IR, despite the equivalent inhibitory effects of KU55933 on DSB repair in both the proficient and the deficient cell lines. P65 siRNA had no effect on DSB repair in either breast cancer cell line. When combined with KU55933, DSB repair was inhibited to the same extent as KU55933 alone in both breast cancer cell lines. P65 siRNA alone sensitised both cell lines to IR. A combination of p65 siRNA and KU55933 resulted in no further sensitisation compared to either one alone. Taken together these data support the hypothesis that KU55933-mediated radio-sensitisation is solely a consequence of its inhibition of NF-κB activation. We conclude that radiotherapy deploying ATM inhibitors may be particularly advantageous in tumours where NF-κB is constitutively activated.

The correlation between personality type and performance time while wearing a respirator.

Many studies have shown that individuals perform better if not wearing a respirator compared with wearing a respirator. This study examined the degree of performance reduction attributable to specific dominant character traits. The subjects performed on a treadmill at a constant speed and grade resulting in 80-85% VO(2)max. A modified M40 respirator was used to create three levels of inspiratory resistance: 2.8, 16.8, and 27.3 cmH(2)O*(sec/L). The 31 subjects were tested using a Myers-Briggs Type Indicator and State-Trait Anxiety Inventory. Multiple regressions and an ANOVA were used to test for correlation. When air intake is very constricted, the only multiple regression equation that was found to be statistically significant was sensing-intuition (how one takes in information) and thinking-feeling (how one makes a decision) vs. performance time for the highest value of inhalation resistance. A simple linear regression between trait anxiety level and performance time was not found to be statistically significant for the same highest value of inhalation resistance.

Effects of novel inhibitors of poly(ADP-ribose) polymerase-1 and the DNA-dependent protein kinase on enzyme activities and DNA repair.

DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1.

The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.

[Delusion and sleep deprivation]. / Bouffée délirante et privation de sommeil.

In this article, the authors report two observations of short delusion that occurred after taking Guronsan--a psychostimulant commercialized in France--for a few days, with the intention of maintaining a total deprivation of sleep for three days in both cases. The ensuing clinical picture included a state of depersonalization, a loss of the sense of reality, illusions and even visual hallucinations as well as a delirious feeling of persecution. These disorders altered with the state of vigilance and the patients remembered them clearly. The authors discussed the etiopathogenic role of this psychotrope, as its components--acid ascorbic, glucuronamide and caffein--are not mentioned in literature as causing factors of a psychotic state. Then they compared this psychotrope with other molecules: amphetamines in particular may start a delirium of persecution, but normally they just reveal an underlying psychotic structure, which doesn't seem to be the case here, where the two young adults were only found a little immature. Chloroquine has sometimes been incriminated for disorders similar to those mentioned above, with a difference lying in a greater stability in the duration of these disorders that would persist several days after the end of the treatment. The clinical picture of the two cases was more labile and sedation was complete as soon as the absorption of the psychotrope was interrupted and sleep was restored at the same time. That is why the authors emphasize the importance of the deprivation of sleep as a causing factor of those delusion disorders which have particularly been observed in the case of solitary navigators. The psychiatrist dealing with emergencies shouldn't overlook this clinical and etiological possibility, all the less so as the treatment is simple and the resort to neuroleptics unnecessary.

Comparison between allograft plus demineralized bone matrix versus autograft in anterior cervical fusion. A prospective multicenter study.

STUDY DESIGN: This study analyzed the fusion results of an allograft-demineralized bone matrix composite versus autograft in a prospective series of patients undergoing surgery for cervical disc disease. OBJECTIVES: To determine the fusion rates of allograft-demineralized bone matrix composite in anterior cervical fusion as compared with the gold standard autograft. SUMMARY OF BACKGROUND DATA: For the anterior cervical fusion, the use of freeze-dried allograft is well documented in the literature, citing its effectiveness and inferior fusion rates. The use of demineralized bone matrix in conjunction with freeze-dried allograft in anterior cervical fusion has not been reported. METHODS: This study was done in a prospective fashion in two medical centers. One group received autograft from the anterior iliac crest, whereas others received freeze-dried allograft augmented with demineralized bone matrix (Grafton, Osteotech, Inc., Shrewsbury, New Jersey). For the autograft group, the standard Smith-Robinson grafting technique was used. For the allograft composite group, demineralized bone matrix was pasted onto the freeze-dried allograft and into the disc space before graft insertion. The autograft group consisted of 38 patients with age ranging 26-71 years (mean, 46.1 years) and follow-up periods of 12-33 months (mean, 18.4 months). There were 19 one-level, 17 two-level, and two three-level fusions. Similarly, the allograft group consisted of 39 patients with age ranging 28-80 years (mean, 48.0 years) with follow-up period of 12-31 months (mean, 17.5 months). There were 19 one-level, 16 two-level, and four three-level fusions. Clinical and radiographic follow-up evaluations were completed at 3-month intervals. Radiographs taken 12 months after surgery were analyzed blindly. RESULTS: Pseudarthrosis developed in 46.2% of patients (33.3% of levels) in the allograft-demineralized bone matrix group compared with 26.3% (22% of levels) in the autograft group (P = 0.11 for patients, P = 0.23 for levels). For patients undergoing two-level fusions, 37.5% of allograft-demineralized bone matrix failed compared with 23.5% of autografts. For single-level fusions, 47.4% of allograft patients developed a pseudarthrosis compared with 26.3% in the autograft group. Graft collapse of > or = 3 mm was noted in 11% of the autograft group versus 19% in the allograft-demineralized bone matrix group (P = 0.32). Graft collapse of > or = 2 mm occurred in 24.4% of autograft patients compared with 39.7% of the allograft-demineralized bone matrix group (P = 0.09). Smokers had an increased rate of pseudarthrosis (47.1%) compared with nonsmokers (27.9%, P = 0.13). CONCLUSIONS: The study revealed that the allograft-demineralized bone matrix construct gives a higher rate of graft collapse and pseudarthrosis when compared with autograft in a prospective series, although the differences were not statistically significant. The pseudarthrosis rate in the series may be high because of the large percentage of smokers and radiographic evaluation techniques. For the purpose of solid radiographic fusion, the use of autograft is recommended in anterior cervical surgery until other acceptable osteoinductive materials are developed.

[The patient with Hansen's disease confronts his distress]. / Le hansénien face à son angoisse.

This article is about the effect of Hansen's disease on the personality of 29 patients of the Institute of Applied Leprology of Dakar, Foundation of the Order of Malta. This approach to the distress of these patients was done by an inquiry based on sociocultural and clinical variables and compared to a study composed of tubercular and psychiatric patients. This study reports four distress levels (loss of identity, loss of object, forlornness, culpability) which are distinguished by sex and age. The actual analysis of the experience of this distress shows the importance of support of the traditional representation of these leprous patients' psychology.

[Various sociocultural findings concerning sexual impotence in Senegal]. / Quelques données socio-culturelles dans l'impuissance sexuelle au Sénégal.

This study emphasizes the role of the socio-cultural status and consequently of psychologic factors in sexual impotence of the black African in Senegal: --role of age, related to the perception of a dual status; --role of the ethnic group giving or not preeminence to sex; --rank in the brotherhood which may raise a conflict with ancestor; --matrimonial situation having significance of an acceptance of the cultural code about sex; --somatizations expressing the sexual potentialities; --non structured neurosis as expression of psychic and sexual inhibition; --religious behaviour with opportunities of conflicts with the religious authorities.

A correlation between clinical pancreatitis and isoenzyme patterns of amylase.

Fifty-seven patients admitted with the clinical diagnosis of acute pancreatitis had isoamylase analysis on their sera to determine the source of their hyperamylasemia. Our objective was to correlate the isoamylase pattern with our clinical observations. Thirty-nine of 57 patients (68%) had pancreatic hyperamylasemia as expected, but 18 of 57 patients (32%) had normal levels of pancreatic amylase. The hyperamylasemia in the latter group was due either to nonpancreatic hyperamylasemia (16 of 57) of macroamylasemia (2 of 57). Consequently, hyperamylasemia associated with abdominal pain, nausea, and vomiting led to the incorrect diagnosis of acute pancreatitis in 32% of the patients. The measurement of isoamylase profiles can be done rapidly and inexpensively. Knowledge that hyperamylasemia is nonpancreatic in origin may have an important influence on treatment, hospitalization, and the extent of laboratory and radiologic investigation.