The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line.

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double strand breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior.

The chromatin-associated 53BP1 ortholog, HSR-9, regulates recombinational repair and X chromosome segregation in the Caenorhabditis elegans germ line.

53BP1 plays a crucial role in regulating DNA damage repair pathway choice and checkpoint signaling in somatic cells; however, its role in meiosis has remained enigmatic. In this study, we demonstrate that the Caenorhabditis elegans ortholog of 53BP1, HSR-9, associates with chromatin in both proliferating and meiotic germ cells. Notably, HSR-9 is enriched on the X chromosome pair in pachytene oogenic germ cells. HSR-9 is also present at kinetochores during both mitotic and meiotic divisions but does not appear to be essential for monitoring microtubule-kinetochore attachments or tension. Using cytological markers of different steps in recombinational repair, we found that HSR-9 influences the processing of a subset of meiotic double strand breaks into COSA-1-marked crossovers. Additionally, HSR-9 plays a role in meiotic X chromosome segregation under conditions where X chromosomes fail to pair, synapse, and recombine. Together, these results highlight that chromatin-associated HSR-9 has both conserved and unique functions in the regulation of meiotic chromosome behavior.

53bp1 mutation enhances brca1 and bard1 embryonic lethality in C. elegans.

In mice, mutation of brca1 results in embryonic lethality, which is partially suppressed by 53bp1 mutation. In contrast, mutation of the C. elegans BRCA1 ortholog, brc-1 , or its binding partner, brd-1 , lead to only mild embryonic lethality. We show that in C. elegans , brc-1 and brd-1 embryonic lethality is enhanced when 53bp1 ortholog, hsr-9 , is also mutated. This is not a consequence of activating polq-1 -dependent microhomology-mediated end joining, as polq-1 mutation does not suppress embryonic lethality of hsr-9 ; brc-1 mutants. Together, these results suggest that BRC-1 - BRD-1 and HSR-9 function in parallel pathways and do not act antagonistically as in mammals.

Inducible degradation of dosage compensation protein DPY-27 facilitates isolation of Caenorhabditis elegans males for molecular and biochemical analyses.

Biological sex affects numerous aspects of biology, yet how sex influences different biological processes have not been extensively studied at the molecular level. Caenorhabditis elegans, with both hermaphrodites (functionally females as adults) and males, is an excellent system to uncover how sex influences physiology. Here, we describe a method to isolate large quantities of C. elegans males by conditionally degrading DPY-27, a component of the dosage compensation complex essential for hermaphrodite, but not male, development. We show that germ cells from males isolated following DPY-27 degradation undergo meiosis and spermiogenesis like wild type and these males are competent to mate and sire viable offspring. We further demonstrate the efficacy of this system by analyzing gene expression and performing affinity pull-downs from male worm extracts.

In Vitro Evaluation of Common Antimicrobial Solutions Used for Breast Implant Soaking and Breast Pocket Irrigation-Part 2: Efficacy Against Biofilm-Associated Bacteria.

BACKGROUND: Biofilm-associated bacteria have been observed in both breast implant revision and tissue expander-implant exchange surgeries. The utilization of antimicrobial solutions in breast surgery, especially those containing triple antibiotics (TAB) and/or 10% povidone-iodine (PI), may help reduce existing biofilm-associated bacteria, which is particularly important in a mature breast pocket that may contain residual bacteria from a previously colonized implant surface or, theoretically, bacteria that may arrive postoperatively through hematogenous spread. OBJECTIVES: A series of in vitro assessments was performed to evaluate the antimicrobial utility of TAB and PI, either alone or in combination, against preformed biofilm-associated bacteria. METHODS: Preformed biofilm-associated gram-positive and gram-negative bacterial strains were exposed to TAB and PI ± TAB for up to 30 minutes in a bacterial time-kill assay. Efficacy of various dilutions of PI and the effects of serum protein on PI efficacy were also investigated. RESULTS: TAB was ineffective at the timeframes tested when utilized alone; when utilized in conjunction with PI, significant log reduction of all biofilm-associated bacterial species tested was achieved when treated for at least 5 minutes. PI alone at a concentration of 25% or higher was also effective, although its efficacy was negatively affected by increasing serum protein concentration only for Staphylococcus epidermidis. CONCLUSIONS: Our data indicate that PI-containing solutions significantly reduce biofilm-associated bacteria, suggesting potential utility for breast pocket irrigation during revision or exchange surgeries. Care should be taken to minimize excessive dilution of PI to maintain efficacy.

In Vitro Evaluation of Common Antimicrobial Solutions Used for Breast Implant Soaking and Breast Pocket Irrigation-Part 1: Efficacy Against Planktonic Bacteria.

BACKGROUND: Planktonic bacteria can be inadvertently introduced during breast surgery procedures, which are hypothesized to lead to complications such as infection, capsular contracture, breast implant-associated anaplastic large cell lymphoma, and a prolonged local inflammatory response. The utilization of antimicrobial solutions such as triple antibiotic solution (TAB) and/or 10% povidone-iodine (PI) in breast pocket irrigation or implant soaking has been proposed to reduce planktonic bacterial attachment and potential complications. OBJECTIVES: A series of in vitro assessments were performed to evaluate the antimicrobial utility of TAB and PI, either alone or in combination, against planktonic bacteria. METHODS: Planktonic gram-positive and gram-negative bacterial strains were exposed to TAB and PI ± TAB for up to 10 minutes in a bacterial time-kill assay. The efficacy of various dilutions of PI as well as the effects of serum protein on PI efficacy were also investigated. RESULTS: TAB was ineffective at the timeframes tested (≤10 minutes) when utilized alone; however, when utilized with PI, significant log reduction of all tested planktonic species was achieved. PI alone was also effective, even including dilute concentrations (eg, 0.5% PI), although the presence of serum proteins required higher concentrations of PI (eg, 2.5%) to eradicate the bacterial load. CONCLUSIONS: Our data suggest PI-containing solutions may be preferred over either saline or TAB without PI for primary breast pocket irrigation and implant soaking in primary breast surgeries as a means to significantly reduce planktonic bacteria. These data provide an impetus for surgeons to re-evaluate the efficacy of TAB solution in these clinical settings.

Meiotic Double-Strand Break Processing and Crossover Patterning Are Regulated in a Sex-Specific Manner by BRCA1-BARD1 in Caenorhabditis elegans.

Meiosis is regulated in a sex-specific manner to produce two distinct gametes, sperm and oocytes, for sexual reproduction. To determine how meiotic recombination is regulated in spermatogenesis, we analyzed the meiotic phenotypes of mutants in the tumor suppressor E3 ubiquitin ligase BRC-1-BRD-1 complex in Caenorhabditis elegans male meiosis. Unlike in mammals, this complex is not required for meiotic sex chromosome inactivation, the process whereby hemizygous sex chromosomes are transcriptionally silenced. Interestingly, brc-1 and brd-1 mutants show meiotic recombination phenotypes that are largely opposing to those previously reported for female meiosis. Fewer meiotic recombination intermediates marked by the recombinase RAD-51 were observed in brc-1 and brd-1 mutants, and the reduction in RAD-51 foci could be suppressed by mutation of nonhomologous-end-joining proteins. Analysis of GFP::RPA-1 revealed fewer foci in the brc-1brd-1 mutant and concentration of BRC-1-BRD-1 to sites of meiotic recombination was dependent on DNA end resection, suggesting that the complex regulates the processing of meiotic double-strand breaks to promote repair by homologous recombination. Further, BRC-1-BRD-1 is important to promote progeny viability when male meiosis is perturbed by mutations that block the pairing and synapsis of different chromosome pairs, although the complex is not required to stabilize the RAD-51 filament as in female meiosis under the same conditions. Analyses of crossover designation and formation revealed that BRC-1-BRD-1 inhibits supernumerary COs when meiosis is perturbed. Together, our findings suggest that BRC-1-BRD-1 regulates different aspects of meiotic recombination in male and female meiosis.

FTIR bio-spectroscopy scattering correction using natural biological characteristics of different cell lines.

Fourier transform infrared (FTIR) spectroscopy is a well-known method of analysis, with various applications, including promising potential for analyzing biological samples. In the bio-spectroscopy of cells, Mie scattering may increase, which then causes spectral distortion, due to the similarity of cell size with the IR medium-wavelength. These changes make the spectrum unreliable. In previous scattering elimination studies, questionable estimations were considered. For instance, all cells were considered as spherical objects or cell size was estimated randomly. In an attempt to provide the best equation based on the natural existence of cells for the FTIR Mie scattering correction, we examined the actual biological data of cells - as opposed to those yielded from mathematical manipulations. So five biological factors: cell size, shape, granularity, circularity, and edge irregularities, for each cell line were considered as factors which cause scattering. For measuring cell size, roundness and edge irregularity, microscopy images were obtained and processed. For evaluating cell line granularity, flow cytometry was used. Finally, by including these factors, an algorithm was designed. To assess the accuracy of the proposed algorithm, the trypsinized cell spectrum was considered as the high scattering spectrum. Cells were also cultured on a MirrIR slide, and their ATR-FTIR spectrum was considered as the minimum scattering spectrum. The algorithm using the abovementioned five characteristics was used for 13 different cell lines, and in some cases the corrected spectrum demonstrated more than 97% resemblance with the ATR spectra of the same cells. A comparison between the results of this algorithm with the Bassan et al. (2017) algorithm for scattering correction that is freely available on the Internet was then conducted on two different cell lines, clearly showing the advantages of our algorithm, in terms of accuracy and precision. Therefore, this method can be viewed as a more suitable solution for scattering correction in cell investigations.

Matrix Topographical Cue-Mediated Myogenic Differentiation of Human Embryonic Stem Cell Derivatives.

Biomaterials varying in physical properties, chemical composition and biofunctionalities can be used as powerful tools to regulate skeletal muscle-specific cellular behaviors, including myogenic differentiation of progenitor cells. Biomaterials with defined topographical cues (e.g., patterned substrates) can mediate cellular alignment of progenitor cells and improve myogenic differentiation. In this study, we employed soft lithography techniques to create substrates with microtopographical cues and used these substrates to study the effect of matrix topographical cues on myogenic differentiation of human embryonic stem cell (hESC)-derived mesodermal progenitor cells expressing platelet-derived growth factor receptor alpha (PDGFRA). Our results show that the majority (>80%) of PDGFRA+ cells on micropatterned polydimethylsiloxane (PDMS) substrates were aligned along the direction of the microgrooves and underwent robust myogenic differentiation compared to those on non-patterned surfaces. Matrix topography-mediated alignment of the mononucleated cells promoted their fusion resulting in mainly (~86%⁻93%) multinucleated myotube formation. Furthermore, when implanted, the cells on the micropatterned substrates showed enhanced in vivo survival (>5⁻7 times) and engraftment (>4⁻6 times) in cardiotoxin-injured tibialis anterior (TA) muscles of NOD/SCID mice compared to cells cultured on corresponding non-patterned substrates.

Impact of cardiac arrest resuscitated donors on heart transplant recipients' outcome.

OBJECTIVE: To evaluate the influence of cardiac arrest-resuscitated donors (CARDs) on the outcome of heart recipients. METHODS: Patients transplanted between July 2004 and December 2012 were divided into 2 groups according to the history of cardiac arrest in donors and their clinical records were retrospectively reviewed. RESULTS: A total of 584 heart transplantations were performed during the study period, and 117 recipients received an organ from a CARD. There were no differences between the 2 groups with regards to recipient age, sex, cardiomyopathy, preoperative extracorporeal membrane oxygenation, national high emergency waiting list, and redo surgery. Donors who sustained a cardiac arrest were significantly younger (44 [32-51] vs 49 [41-56] years; P < .001), their main cause of death was anoxia (57% vs 1%; P < .001), and they had significantly greater troponin T peak levels (0.51 [0.128-3.108] vs 0.11 [0.04-0.43] ng/mL; P < .001). Median cardiac arrest duration was 15 minutes (5-25). No difference was noted in donors with regards to left ventricular ejection fraction at time of organ procurement (62% ± 8% vs 63% ± 8%; P = .2). There were no differences between the 2 groups with regards to ischemic time (179 ± 60 vs 183 ± 59 minutes; P = .43), need for postoperative extracorporeal membrane oxygenation for primary graft failure (31% vs 30%; P = .993) and 30-days mortality. Recipients receiving an organ from a CARD had a significantly better 10 year survival (69.4% vs 50.4%; P = .017). CONCLUSIONS: History of cardiac arrest in donors with a preserved left ventricular ejection fraction at time of organ procurement doesn't affect outcome of heart recipients.