Functional assay for BRCA1: mutagenesis of C-terminal region reveals critical residues for transcription activation

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor but its precise biochemical function remains unknown. The BRCA1 C-terminus acts as a transcription activation domain and germ-line cancer-predisposing mutations in this region abolish transcription activation whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 C-terminus (aa 1560–1863) consists of two BRCT (BRCA1 C-terminal) domains preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis followed by a functional transcription assay in yeast. First, error prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat and human BRCA1. Second, random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions N-terminal to the BRCT domains retained wild-type activity whereas insertions in the BRCT domains were, with few exceptions, deleterious. Third, site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the C-terminus. Deletion analysis revealed that the integrity of the most C-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay

Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor but its precise biochemical function remains unknown. The BRCA1 C-terminus acts as a transcription activation domain and germ-line cancer-predisposing mutations in this region abolish transcription activation whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 C-terminus (aa 1560–1863) consists of two BRCT (BRCA1 C-terminal) domains preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis followed by a functional transcription assay in yeast. First, error prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat and human BRCA1. Second, random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions N-terminal to the BRCT domains retained wild-type activity whereas insertions in the BRCT domains were, with few exceptions, deleterious. Third, site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the C-terminus. Deletion analysis revealed that the integrity of the most C-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay

Redox-Triggered Infection by Disulfide-Shackled Human Immunodeficiency Virus Type 1 Pseudovirions

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant’s infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process

Nutritional Knowledge and Dietary Habits of Student-Athletes in a State University: Towards a Contextualized Nutrition Education Plan

Sustainable Development Goal 2 aims to end hunger, achieve food security, improve nutrition, and promote healthy and sustainable diets. Student-athletes deal with unique challenges due to the demands of physical and mental requirements of their sports, making proper nutrition and dietary habits crucial for their overall health and performance. Therefore, this study aimed to analyze the relationship between nutritional knowledge and dietary habits among State Colleges and Universities Athletic Association (SCUAA) Athletes at a state university in Pampanga, Philippines, as a basis for the development of a contextualized Nutrition Education Plan (NEP). The study was grounded on a postpositivist philosophical worldview that employed a quantitative non-experimental approach, specifically a correlational design. A total of 177 student-athletes were assessed for their nutritional knowledge and dietary habits using survey questionnaires. It was determined that student-athletes’ level of nutritional knowledge was described as “knowledgeable” while their perceived dietary habits were described as “good”, but still, there is an area for continued improvement. Meanwhile, it was found that there was no significant difference between nutritional knowledge and dietary habits of student-athletes when grouped according to their demographic profile. On the other hand, there was a significant but weak positive relationship between nutritional knowledge and dietary habits of student-athletes. The results of student-athletes’ nutritional knowledge and dietary habits paved the way for a contextualized NEP anchored on the student-athlete. The study recommends generalizing the NEP and considering the other athletes who also need nutrition and dietary habits improvement

Domain of functional affectation and profile of children with special needs in the 4th district of Cavite (Research locale set in selected barangays in Dasmarinas City, Cavite)

This study used total enumeration technique and snowball or referral technique to gather data of children with special needs. Questionnaires or survey forms were the data gathering instruments. The questions that were asked in the survey aimed to identify the demographic, educational, socioeconomic and rehabilitative services profile of children with special needs. The survey also included the non-monetary support system, wherein the researchers asked if the participants (CWD) were receiving any non-financial help from the government and rehabilitative services if given. Frequency distribution and percentage were used to determine the general trends. This study concludes that most of children with special needs in the 4th district of Cavite within the selected barangays were males, currently studying and belong to Class E economic status. Most of them were receiving highly satisfactory psychosocial and non-monetary support. Most of the respondents were assessed by medical doctors but were not able to receive the results/diagnosis. Majority of children with special needs were identified to have speech, language, and communication problems. The researchers concluded that these children with special needs are least prioritized in terms of medical/rehabilitative and educational services

Development of quantitative precipitation estimation (QPE) relations for dual-polarization radars based on raindrop size distribution measurements in Metro Manila, Philippines

Abstract Quantitative precipitation estimates (QPE) can be further improved using estimation algorithms derived from localized raindrop size distribution (DSD) observations. In this study, DSD measurements from two disdrometer stations within Metro Manila during the Southwest monsoon (SWM) period were used to investigate the microphysical properties of rainfall and develop localized dual-polarimetric relations for different radar bands and rainfall types. Observations show that the DSD in Metro Manila is more distributed to larger diameters compared to Southern Luzon and neighboring countries and regions in the Western Pacific. This is reflected by the relatively higher mass-weighted mean diameter (D m ) and smaller shape (μ) and slope (Λ) parameters measured in the region. The average values of D m and normalized intercept parameter (N w ) in convective rain samples also suggest that convective rains in Metro Manila are highly influenced by both continental and oceanic convective processes. Dual-polarimetric variables simulated using the T-matrix scattering method showed good agreement with disdrometer-derived reflectivity (Z H ) values. The 0.5 dB and 0.3° km−1 thresholds for the differential reflectivity (Z DR ) and specific differential phase (K DP ) based on the blended algorithm of Cifelli (J Atmos Ocean Technol 28:352-364, 2011) and Thompson et al. (2017) are proven to be useful since the utility of the dual-polarimetric variables as rainfall estimators are shown to have dependencies on the radar band and rainfall type. Evaluation of the QPE products with respect to the C-band shows that R (K DP , Z DR ) has the best performance among the dual-pol relations and statistically outperformed the conventional Marshall & Palmer relation [R(Z MP )]. The results show that dual-polarimetric variables such as ZDR and KDP can better represent the DSD properties compared to one-dimensional Z, hence providing more accurate QPE products than the conventional R(Z) relations

Predictors of weight loss in obese patients with obstructive sleep apnea

Vol. 26 pp 753-762Purpose: Consistent predictors of weight loss outcomes with very low-energy diets (VLEDs) in obstructive sleep apnea (OSA) have not been identified. This study aimed to identify variables predictive of weight loss success in obese patients with OSA undertaking an intensive weight loss programme. Methods: We analysed biological, psychological, and behavioural variables as potential predictors of weight loss in obese patients with OSA after a 2-month VLED followed by one of two 10-month weight loss maintenance diets. Actigraphy, in-lab polysomnography, urinary catecholamines, and various psychological and behavioural variables were measured at baseline, 2, and 12 months. Spearman’s correlations analysed baseline variables with 2-month weight loss, and 2-month variables with 2–12 month-weight change. Results: Forty-two patients completed the VLED and thirty-eight completed the maintenance diets. Actigraphy data revealed that late bedtime (rs =  − 0.45, p =  < 0.01) was correlated with 2-month weight loss. The change in the time that participants got out of bed (rise-time) from baseline to two months was also correlated with 2-month weight loss (rs = 0.36, p = 0.03). The Impact of Weight on Quality of Life-Lite questionnaire (IWQOL) Public Distress domain (rs =  − 0.54, p =  < 0.01) and total (rs =  − 0.38, p = 0.02) scores were correlated with weight loss maintenance from 2 to 12 months. Conclusions: Results from this small patient sample reveal correlations between actigraphy characteristics and weight loss in obese patients with OSA. We suggest the IWQOL may also be a useful clinical tool to identify OSA patients at risk of weight regain after initial weight loss.Thomas J. Altree, Delwyn J. Bartlett, Nathaniel S. Marshall, Camilla M. Hoyos, Craig L. Phillips, Callum Birks, Aran Kanagaratnam, Anna Mullins, Yasmina Serinel, Keith K.H. Wong, Brendon J. Yee, Ronald R. Grunstein, Elizabeth A. Cayana

HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5

The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruse