The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51.

The breast cancer susceptibility protein, BRCA2, is essential for recombinational DNA repair. BRCA2 delivers RAD51 to double-stranded DNA (dsDNA) breaks through interaction with eight conserved, approximately 35 amino acid motifs, the BRC repeats. Here we show that the solitary BRC4 promotes assembly of RAD51 onto single-stranded DNA (ssDNA), but not dsDNA, to stimulate DNA strand exchange. BRC4 acts by blocking ATP hydrolysis and thereby maintaining the active ATP-bound form of the RAD51-ssDNA filament. Single-molecule visualization shows that BRC4 does not disassemble RAD51-dsDNA filaments but rather blocks nucleation of RAD51 onto dsDNA. Furthermore, this behavior is manifested by a domain of BRCA2 comprising all eight BRC repeats. These results establish that the BRC repeats modulate RAD51-DNA interaction in two opposing but functionally reinforcing ways: targeting active RAD51 to ssDNA and prohibiting RAD51 nucleation onto dsDNA. Thus, BRCA2 recruits RAD51 to DNA breaks and, we propose, the BRC repeats regulate DNA-binding selectivity.

Effect of random fiber networks on bubble growth in gelatin hydrogels.

In hydrodynamics, the event of dynamic bubble growth in a pure liquid under tensile pressure is known as cavitation. The same event can also be observed in soft materials (e.g., elastomers and hydrogels). However, for soft materials, bubble/cavity growth is either defined as cavitation if the bubble growth is elastic and reversible or as fracture if the cavity growth is by material failure and irreversible. In any way, bubble growth can cause damage to soft materials (e.g., tissue) by inducing high strain and strain-rate deformation. Additionally, a high-strength pressure wave is generated upon the collapse of the bubble. Therefore, it is crucial to identify the critical condition of spontaneous bubble growth in soft materials. Experimental and theoretical observations have agreed that the onset of bubble growth in soft materials requires higher tensile pressure than pure water. The extra tensile pressure is required since the cavitating bubble needs to overcome the elastic and surface energy in soft materials. In this manuscript, we developed two models to study and quantify the extra tensile pressure for different gelatin concentrations. Both the models are then compared with the existing cavitation onset criteria of rubber-like materials. Validation is done with the experimental results of threshold tensile pressure for different gelatin concentrations. Both models can moderately predict the extra tensile pressure within the intermediate range of gelatin concentrations (3-7% [w/v]). For low concentration (∼1%), the network's non-affinity plays a significant role and must be incorporated. On the other hand, for higher concentrations (∼10%), the entropic deformation dominates, and the strain energy formulation is not adequate.

High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy.

Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.

Effects of the Melatonin and Vitamin E (Alpha-Tocopherol Acetate) as Antioxidants on Biochemical Blood Parameters, Lipid Profile, and Muscle Vitamin E Concentration in Awassi Lambs Fed a High-Energy Diet and Normal Diet.

Health specialists currently suggest low-cholesterol diets, suggesting that cholesterol in the form of high-density lipoprotein (HDL) reduces the risk of chronic atherosclerosis. The large volume of literature describes the biological roles of vitamin E and its application to preventing disease and improving the health and productive performances of farm animals. The present study aimed to evaluate the effects of vitamin E (Alpha-tocopherol acetate) supplementation and melatonin implants on biochemical blood, lipid profile and muscle vitamin E content of Awassi male lambs fed by a high and normal diet in Iraq. The lambs were divided into teen groups as control normal energy diet T1 (NED) T2 (HED) concentrated lamb fattening feed. Two levels of melatonin (18 and 36 mg implant) were applied to T3, T4, T5, and T6 treatment and 2 levels of Vitamin E (Alpha-tocopherol acetate) diet 200 mg/kg, 400 mg/kg to T7. T8. T9 and T10, respectively. Results from the present study indicate that Vitamin E 200, 400 mg/lamb/day and melatonin implantation 18 mg, 36 mg/lamb/day significantly (P<0.05) increased total protein in serum while decreasing globulin level, glucose concentration in serum, melatonin implantation 36 mg/lamb and vitamin E 400 mg/lamb/day recorded significantly (P<0.05). The same effect on decreasing cholesterol concentration in serum 42.6mg\dl, 40.5 mg\dl, respectively, compared to non-treated groups. Vitamin E 200 mg/kg/lamb recorded the lowest AST level in serum, 43.3. Lambs implanted with Melatonin 36 mg/lamb and fed a high-energy diet (T8) resulted in a significant decrease of serum ALT activity (P<0.05) in comparison to other treated groups 12.7 U/L was achieved. Lambs fed a normal energy diet with vitamin E 200 mg/kg/lamb (T4) exceeded other treated groups, decreasing ALT serum levels by 9.35 U/L. Interestingly, muscle vitamin E concentrations for lambs received 200, 400 mg/lamb/day on the 2nd, 7th and 14th days of the storage period, and fed high energy diet (T10) or normal energy diet (T5) were significantly higher compared to control group (T1, T6).

Heat transport in inclined flow towards a rotating disk under MHD.

Flow towards a rotating disk is of highly practical significance in numerous engineering applications such as Turbine disks, rotary type machine systems and many more. In light of this, the current work is an attempt to explore MHD oblique flow towards a rotating disk. Hydromagnetic effects in addition to heat transfer is taken into consideration. The flow governing Partial Differential Equations are altered to a system to coupled non-linear Ordinary Differential Equations through scaling group of transformations which afterwards are tackled using Shooting Algorithm. The impact of obliqueness parameter γ, rotation ratio parameter [Formula: see text] and magnetic field parameter M on 2-dimensional and 3-dimensional stream contours are presented. Location of the shear center varies with magnetic field parameter. Heat flow at the disk surface boosts with magnet field parameter M and rotation ratio parameter [Formula: see text].

The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange.

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2([BRC1-8])). Here, we show that BRCA2([BRC1-8]) exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2([BRC1-8]) alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2([BRC1-8]) promotes RAD51 loading onto ssDNA. In contrast, BRCA2([BRC1-8]) has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2([BRC1-8]) stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing recombination products. Collectively, our findings suggest that BRCA2([BRC1-8]) targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.

Influence of Trichoderma harzianum and Bacillus thuringiensis with reducing rates of NPK on growth, physiology, and fruit quality of Citrus aurantifolia.

Continuous use of chemical fertilizers gradually shrinks the crop yield and quality, and these adverse effects can be reduced by adopting new sustainable practices such as the use of manure, biofertilizers, and nano fertilizers. Limited information is existed on the application of Trichoderma harzianum and Bacillus thuringiensis microbes to improve lemon seedlings growth, physiology, and fruit formation. Therefore, the current study is aimed to evaluate the effects of T. harzianum and B. thuringiensis microbes mixing with low levels of inorganic fertilizer (NPK) on the plant growth, development, and quality of limau nipis (key lemon) fruits. The lemon seedlings growing media were inoculated during transplanting with T. harzianum and B. thuringiensis at various NPK fertilizers under polybagged conditions. The seedlings were grown around eighteen (18) months after inoculation with biofertilizers followed by Randomized Complete Block Design (RCBD) with five (5) replications. The results showed that T. harzianum with 50 g of NPK treatment (T2) increased the seedling's height, branch number, leaf height, ground area, and absolute growth rate (AGR) plant height by 50.12%, 107.84%, 17.91%, 17.91%, 116.93%, and 56.02%, respectively, over the control treatment. The number of leaves (60.82%), leaf area (42.75%), stem diameter (27.34%), specific leaf area (SLA) (39.07%), leaf area index (LAI) (54.40%), and absolute growth rate for leaf number (73.86%), leaf area (306.85%) and stem diameter (46.8%) of lemon seedlings increased significantly with B. thuringiensis plus 50 g NPK treatment (T3). The applications of B. thuringiensis with 25 g NPK fertilizer treatment (T5) increased leaf fresh weight (LFW), leaf dry weight (LDW), leaf moisture content (LMC), specific leaf weight (SLW), leaf relative growth rate (RGR), and chlorophyll content by 96.45%, 56.78%, 13.60%, 24.76%, 45.45%, and 16.22%, respectively, over the control group. In addition, T5 treatment increased the fruits number, individual fruit weight, fruit diameter, fruit dimension, leaf total soluble solids (TSS), and fruit TSS content of lemon tress by 81.81%, 55.52%, 43.54%, 25.69%, 89.47%, and 70.78% compared to the control treatment. Furthermore, soil inoculation of B. thuringiensis significantly increased the pulp to peel ratio and juice content of lemon fruits. From this study, it can be concluded that soil inoculation of both T. harzianum and B. thuringiensis with 25-50% NPK during transplanting improved plant growth, physiology, and fruit quality of limau nipis trees.

Incorporating non-stomatal limitation improves the performance of leaf and canopy models at high vapour pressure deficit.

Vapour pressure deficit (D) is projected to increase in the future as temperature rises. In response to increased D, stomatal conductance (gs) and photosynthesis (A) are reduced, which may result in significant reductions in terrestrial carbon, water and energy fluxes. It is thus important for gas exchange models to capture the observed responses of gs and A with increasing D. We tested a series of coupled A-gs models against leaf gas exchange measurements from the Cumberland Plain Woodland (Australia), where D regularly exceeds 2 kPa and can reach 8 kPa in summer. Two commonly used A-gs models were not able to capture the observed decrease in A and gs with increasing D at the leaf scale. To explain this decrease in A and gs, two alternative hypotheses were tested: hydraulic limitation (i.e., plants reduce gs and/or A due to insufficient water supply) and non-stomatal limitation (i.e., downregulation of photosynthetic capacity). We found that the model that incorporated a non-stomatal limitation captured the observations with high fidelity and required the fewest number of parameters. Whilst the model incorporating hydraulic limitation captured the observed A and gs, it did so via a physical mechanism that is incorrect. We then incorporated a non-stomatal limitation into the stand model, MAESPA, to examine its impact on canopy transpiration and gross primary production. Accounting for a non-stomatal limitation reduced the predicted transpiration by ~19%, improving the correspondence with sap flow measurements, and gross primary production by ~14%. Given the projected global increases in D associated with future warming, these findings suggest that models may need to incorporate non-stomatal limitation to accurately simulate A and gs in the future with high D. Further data on non-stomatal limitation at high D should be a priority, in order to determine the generality of our results and develop a widely applicable model.

A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange.

Human BRCA2, a breast and ovarian cancer suppressor, binds to the DNA recombinase RAD51 through eight conserved BRC repeats, motifs of approximately 30 residues, dispersed across a large region of the protein. BRCA2 is essential for homologous recombination in vivo, but isolated BRC repeat peptides can prevent the assembly of RAD51 into active nucleoprotein filaments in vitro, suggesting a model in which BRCA2 sequesters RAD51 in undamaged cells, and promotes recombinase function after DNA damage. How BRCA2 might fulfill these dual functions is unclear. We have purified a fragment of human BRCA2 (BRCA2(BRC1-8)) with 1127 residues spanning all 8 BRC repeats but excluding the C-terminal DNA-binding domain (BRCA2(CTD)). BRCA2(BRC1-8) binds RAD51 nucleoprotein filaments in a ternary complex, indicating it may organize RAD51 on DNA. Human RAD51 is relatively ineffective in vitro at strand exchange between homologous DNA molecules unless non-physiological ions like NH4+ are present. In an ionic milieu more typical of the mammalian nucleus, BRCA2(BRCI-8) stimulates RAD51-mediated strand exchange, suggesting it may be an essential co-factor in vivo. Thus, the human BRC repeats, embedded within their surronding sequences as an eight-repeat unit, mediate homologous recombination independent of the BRCA2(CTD) through a previously unrecognized role in control of RAD51 activity.

BRCA2 Regulates Transcription Elongation by RNA Polymerase II to Prevent R-Loop Accumulation.

The controlled release of RNA polymerase II (RNAPII) from promoter-proximal pausing (PPP) sites is critical for transcription elongation in metazoans. We show that the human tumor suppressor BRCA2 interacts with RNAPII to regulate PPP release, thereby preventing unscheduled RNA-DNA hybrids (R-loops) implicated in genomic instability and carcinogenesis. BRCA2 inactivation by depletion or cancer-causing mutations instigates RNAPII accumulation and R-loop accrual at PPP sites in actively transcribed genes, accompanied by γH2AX formation marking DNA breakage, which is reduced by ERCC4 endonuclease depletion. BRCA2 inactivation decreases RNAPII-associated factor 1 (PAF1) recruitment (which normally promotes RNAPII release) and diminishes H2B Lys120 ubiquitination, impeding nascent RNA synthesis. PAF1 depletion phenocopies, while its overexpression ameliorates, R-loop accumulation after BRCA2 inactivation. Thus, an unrecognized role for BRCA2 in the transition from promoter-proximal pausing to productive elongation via augmented PAF1 recruitment to RNAPII is subverted by disease-causing mutations, provoking R-loop-mediated DNA breakage in BRCA2-deficient cells.

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage.

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

Assaying for the dual incisions of nucleotide excision repair using DNA with a lesion at a specific site.

Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily specifically measured by detecting the 24-32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern blot hybridization. The second method is to 3'- end-label the excised oligonucleotide directly with radiolabeled dNTP and a DNA polymerase, using a complementary oligonucleotide with a 5'-overhang that serves as a template. This protocol is fast and sensitive, but relies on accurate foreknowledge of the site of 3'-incision for the particular lesion being used.

DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2.

Germline mutations affecting a single allele of BRCA2 increase susceptibility to breast and ovarian cancer, whilst germline inheritance of certain bi-allelic mutations causes a Fanconi anaemia-like syndrome. Here, we review current knowledge of the BRCA2 protein, focussing on recent studies that provide mechanistic insight into its biological function in regulating DNA recombination reactions mediated by the RAD51 recombinase. We argue that the chromosomal instability and cancer predisposition provoked by BRCA2 inactivation are a consequence of the failure to re-start stalled DNA replication, and to repair DNA double-strand breaks, through error-free pathways that depend on homologous pairing between DNA strands.

Telemedicine: caring for patients across boundaries.

Telemedicine combines computer, video and telecommunications to provide healthcare to patients at distant sites. With the improved camera and transmission technologies of the 1990s, telemedicine can be used in a variety of situations. There are two basic technological systems: live interactive video and still image ("store and forward"). Potential users include patients who live in rural or difficult to reach geographic areas, who are confined (i.e. prison inmates), Telemedicine can allow ambulatory patients to continue living at home rather than moving into costly nursing facilities. Home telemedicine also allows greater responsiveness and higher frequency of visits by home care nurses, potentially reducing future hospital visits and costs. Two home telemedicine models are the personal telemedicine unit and the enhanced personal telemedicine module with pc-based video. Telemedicine technologies developed by the military for use on the battlefield that could be adapted for civilian use include medical simulations, individual monitoring devices and biosensors, portable retinal display monitors, life support for trauma/transport, and diagnostic ultrasound imagery. Ultimately, the benefits of telemedicine will be consistency of care, easy access to specialized consultants, higher responsiveness to patient needs, and lower overall healthcare costs.

The personal telemedicine system. A new tool for the delivery of health care.

An inexpensive home telemedicine system has been developed, comprising a personal telemedicine unit in the patient's home connected by ordinary telephone lines to a central nursing station. Using this system a nurse or other health-care professional at the central station was able to make a 'video visit' to the patient's home. In a preliminary trial, patients were referred by the patient's physician or by a home-care nurse. All 12 patients learned to use the personal telemedicine unit easily and effectively, and there were no complications related to its use. A significant finding was a reduction in the number of home-care visits in seven of the 12 patients (58%). Telecare using the personal telemedicine system was significantly cheaper than care delivered by conventional routes. The average charge was about $15 for a video visit by a nurse, compared with about $90 for a real visit.

Home heparin infusion in the management of deep vein thrombophlebitis.

Feasibility of home Heparin infusion in the management of deep vein thrombophlebitis (DVT) was assessed in 15 patients. Patients with chest pain, dyspnea, bleeding tendencies, immediate postoperative state, and lack of reliable care giver were excluded. Five patients received the entire course of Heparin infusion at home and 10 were initially started on Heparin in the hospital. Regular assessment and monitoring of blood coagulation parameters were performed by a visiting home nurse clinician in consultation with the attending physician. Eleven physicians were involved in this management and made a total of six physician home visits. Results showed improvement in all but one patient, who had advanced malignancy and resistant thrombophlebitis and who eventually died. No complications occurred among the patients studied. Patient and family surveys indicated a high degree of satisfaction and preference for this modality of care. Analysis of cost indicated a 48% savings compared with similar treatment administered in a hospital setting.

Telemedicine--a new idea for home care.

Telemedicine could prove to be an important tool in the delivery of home care. Early experience indicates that it keeps patients home, reducing emergency room visits, rehospitalization, and institutionalization. How does it work?

Home chemotherapy. A mobile delivery system.

CareVan Medical Systems specializes in home chemotherapy, differing from other services in its physician involvement. CareVan provides increasing services and advantages to cancer patients receiving treatment at home.